Long-acting adrenomedullin derivative

ABSTRACT

The invention provides a novel adrenomedullin derivative sustainable for a long period which is capable of substantially suppressing unwanted side effects while maintaining pharmacological effects of adrenomedullin. In an exemplary embodiment, the invention relates to a compound represented by formula (I): A-CH 2 —B (I) [wherein A is a modifying group comprising one or more polyethylene glycol groups, and B is a peptide moiety derived from adrenomedullin or a modified form thereof with adrenomedullin activity, wherein the peptide moiety B is linked to the other moieties through a covalent bond of the nitrogen atom of the N-terminal α-amino group of the peptide moiety B to the carbon atom of the methylene group] or a salt thereof, or a hydrate thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application filed under 35 USC 371of PCT/JP2016/077543, filed Sep. 16, 2016, which claims the benefit ofJapanese Patent Application No. 2015-184685, filed Sep. 18, 2015, eachof which is incorporated herein, in its entirety, by reference.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 11924400128SequenceListing. The size of the textfile is 17 KB, and the text file was created on Mar. 14, 2018.

TECHNICAL FIELD

The invention relates to long-acting adrenomedullin derivatives.

BACKGROUND ART

Adrenomedullin (hereinafter, also described as “AM”) is a bioactivepeptide which was isolated and identified from pheochromocytoma in 1993(Non Patent Literature 1). At the beginning of the discovery, AM wasfound to exert a strong vasodilatory hypotensive effect. For example,Patent Literature 1 describes a peptide having a blood pressure-loweringeffect that comprises the amino acid sequence of human AM.

Subsequent studies revealed that AM exerts diverse pharmacologicaleffects such as a cardiovascular protective effect, an anti-inflammatoryeffect, an angiogenic effect, and a tissue repair promoting effect. Inan effort to apply the pharmacological effects of AM to treatment ofdisease, administration of AM to patients with different diseases hasbeen attempted. AM is expected to be useful as a therapeutic agent forinflammatory bowel diseases, pulmonary hypertension, peripheral vasculardiseases, or acute myocardial infarction, among others.

For example, Patent Literature 2 describes an agent for preventing ortreating nonbacterial inflammatory bowel diseases, wherein the agentcomprises, as an active ingredient, adrenomedullin or a derivativethereof that has an activity to suppress nonbacterial inflammation, or asalt thereof that has an activity to suppress nonbacterial inflammation.

Patent Literature 3 describes a method for preventing or treating aninflammatory bowel disease for which the use of a steroid preparation,an immunosuppressant, or a biological product is difficult orinsufficiently effective in a patient in need of prevention or treatmentof the inflammatory bowel disease, the method comprising administeringan effective amount of adrenomedullin, a modified form thereof having anactivity of suppressing inflammation, or a salt of the adrenomedullin orthe modified form having an activity of suppressing inflammation, to thepatient.

Structure-activity relationship studies of AM have advancedidentification of essential sequences that can contribute bioactivity ofAM (Non Patent Literatures 2 to 9).

Peptides are generally known to have a short half-life due to ametabolism in a living body (such as in blood). Therefore, in the caseof using peptides as active ingredients in medicaments, forms of peptidederivatives in which other groups are linked to the peptides can prolonghalf-life in a living body and improve pharmacokinetics, in some cases.

For example, Patent Literature 4 describes a biologically activeintermedin peptide or adrenomedullin peptide characterized by having aserum half-life exceeding 1.5 hours. The literature states that an alkylgroup and a peptide moiety are linked via an amide bond.

Patent Literature 5 describes an AM derivative linked to a polyethyleneglycol (hereinafter, also described as “PEG”) group via the phenolichydroxy group of Tyr¹ of AM.

Patent Literature 6 describes a method comprising reacting PEG-aldehydewith a free amino group of a peptide to produce a peptide derivativehaving the PEG group linked to the free amino group of the peptide. Theliterature describes AM as the peptide.

Non Patent Literature 10 describes an AM derivative in which a PEG groupis linked to the N-terminal α-amino group of AM via an amide bond. Theliterature states that blood half-life of the AM derivative having thelinked PEG group was prolonged.

CITATION LIST Patent Literature

-   Patent Literature 1: JP Patent No. 2774769-   Patent Literature 2: JP Patent No. 4830093-   Patent Literature 3: International Publication No. WO 2012/096411-   Patent Literature 4: International Publication No. WO 2012/138867-   Patent Literature 5: International Publication No. WO 2013/064508-   Patent Literature 6: U.S. Patent Publication. No. US 2009/0252703

Non Patent Literature

-   Non Patent Literature 1: Kitamura K, Kangawa K, Kawamoto M, Ichiki    Y, Nakamura S, Matsuo H, Eto T. Adrenomedullin: a novel hypotensive    peptide isolated from human pheochromocytoma. Biochem Biophys Res    Commun, 30 Apr. 1993, Volume 192, Issue 2, pp. 553-560.-   Non Patent Literature 2: Belloni, A. S. et al., Structure-activity    relationships of adrenomedullin in the adrenal gland. Endocr Res,    1998, Volume 24, Issue 3-4, p. 729-30.-   Non Patent Literature 3: Champion, H. C. et al., Catecholamine    release mediates pressor effects of adrenomedullin-(15-22) in the    rat. Hypertension, 1996, Volume 28, Issue 6, p. 1041-6.-   Non Patent Literature 4: Champion, H. C., G. G. Nussdorfer,    and P. J. Kadowitz, Structure-activity relationships of    adrenomedullin in the circulation and adrenal gland. Regul Pept,    1999, Volume 85, Issue 1, p. 1-8.-   Non Patent Literature 5: Eguchi, S. et al., Structure-activity    relationship of adrenomedullin, a novel vasodilatory peptide, in    cultured rat vascular smooth muscle cells. Endocrinology, 1994,    Volume 135, Issue 6, p. 2454-8.-   Non Patent Literature 6: Garcia, M. A. et al., Synthesis, biological    evaluation, and three-dimensional quantitative structure-activity    relationship study of small-molecule positive modulators of    adrenomedullin. J Med Chem, 2005, Volume 48, Issue 12, p. 4068-75.-   Non Patent Literature 7: Mitsuda, Y. et al., Large-scale production    of functional human adrenomedullin: expression, cleavage, amidation,    and purification. Protein Expr Purif, 2002, Volume 25, Issue 3, p.    448-55.-   Non Patent Literature 8: Roldos, V. et al., Small-molecule negative    modulators of adrenomedullin: design, synthesis, and 3D-QSAR study.    ChemMedChem, 2008, Volume 3, Issue 9, p. 1345-55.-   Non Patent Literature 9: Watanabe, T. X. et al., Vasopressor    activities of N-terminal fragments of adrenomedullin in anesthetized    rat. Biochem Biophys Res Commun, 1996, Volume 219, Issue 1, p.    59-63.-   Non Patent Literature 10: Kubo, K et al., Biological properties of    adrenomedullin conjugated with polyethylene glycol. Peptides, 2014,    Volume 57, p. 118-21.-   Non Patent Literature 11: Kato, J., Kitamura, K. Bench-to-bedside    pharmacology of adrenomedullin. European Journal of Pharmacology,    2015, Volume 764, p. 140-148.

SUMMARY OF INVENTION Technical Problem

As described above, AM derivatives in which other groups such as a PEGgroup are linked to AM are known in order to improve thepharmacokinetics of AM from the viewpoint of improvement insustainability in a living body. However, known AM derivatives aresusceptible to improvement. For example, in the case of linking arelatively large group such as a PEG group to a relatively small peptidesuch as AM, various properties of the resulting AM derivative may varylargely depending on the molecular weight of the PEG group. As describedin Patent Literatures 4 and 5 and Non Patent Literature 10, when apeptide moiety and other groups are linked through a bond which may becleaved by a biological reaction, such as an amide bond or an esterbond, the bond may be cleaved in a relatively short time afteradministration. As in the AM derivative described in Patent Literature5, in the case of linking other groups to the side chain of an aminoacid residue of AM, the conformation of the AM moiety may be changed toreduce the affinity for an AM receptor recognizing AM. In such a case,pharmacological effects as AM of the resulting AM derivative may bereduced.

AM has a strong vasodilatory effect, in addition to pharmacologicaleffects such as a cardiovascular protective effect, an anti-inflammatoryeffect, an angiogenic effect, and a tissue repair promoting effect. Thisstrong vasodilatory effect may cause unwanted side effects such asexcessive decreased blood pressure when AM or an AM derivative isadministered to subjects. The occurrence of such side effects may becomea problem when AM or an AM derivative is used, particularly, in theexpectation that pharmacological effects other than a vasodilatoryeffect are exerted.

The invention, therefore, is intended to provide novel adrenomedullinderivatives sustainable for a long period which are capable ofsubstantially suppressing unwanted side effects while maintainingpharmacological effects of adrenomedullin.

Solution to Problem

The present inventors conducted various investigations of means to solvethe problems described above. The present inventors have found thatlinking of the N-terminal α-amino group of adrenomedullin to a PEG groupwith a specific molecular weight via a methylene group or a urethanegroup can prolong blood half-life of the resulting adrenomedullinderivative as compared to adrenomedullin while retaining bioactivity atthe same level as in adrenomedullin. Furthermore, the present inventorshave found that novel adrenomedullin derivatives having the propertiesdescribed above are capable of substantially suppressing unwanted sideeffects such as excessive decreased blood pressure. The presentinventors have achieved the invention based on the finding describedabove.

That is to say, a summary of the invention is as the following:

(1) A compound represented by formula (I):A-CH₂—B  (I)whereinA is a modifying group comprising one or more polyethylene glycolgroups, andB is a peptide moiety derived from adrenomedullin or a modified formthereof with adrenomedullin activity,wherein the peptide moiety B is linked to the other moieties through acovalent bond of the nitrogen atom of the N-terminal α-amino group ofthe peptide moiety B to the carbon atom of the methylene group,or a salt thereof, or a hydrate thereof.

(2) The compound according to the embodiment (1), wherein A is amodifying group represented by the following formula (II):

whereina is an integer of 1 or larger,m is an integer of 1 or larger,L¹ is a m+1-valent linear or branched linking group, wherein when aplurality of L¹ are present, the plurality of L¹ are the same as ordifferent from each other,L² and L^(2′) are each independently a bond or a divalent linking group,wherein when a plurality of L^(2′) are present, the plurality of L^(2′)are the same as or different from each other,M¹ is a polyethylene glycol group represented by formula (III):^(#)—(CH₂CH₂O)_(n)—**  (III)whereinn is an integer of 1 or larger,** is a binding position to L¹, and# is a binding position to O or L^(2′),wherein when a plurality of M¹ are present, the plurality of M¹ are thesame as or different from each other,M² is a bond or a polyethylene glycol group represented by formula(III), wherein when a plurality of M² are present, the plurality of M²are the same as or different from each other,R¹ is hydrogen, substituted or unsubstituted C₁-C₂₀ alkyl, substitutedor unsubstituted C₂-C₂₀ alkenyl, substituted or unsubstituted C₂-C₂₀alkynyl, substituted or unsubstituted C₃-C₂₀ cycloalkyl, substituted orunsubstituted C₄-C₂₀ cycloalkenyl, substituted or unsubstituted C₄-C₂₀cycloalkynyl, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl, substituted or unsubstituted C₇-C₂₀ cycloalkylalkyl,substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C₁-C₂₀alkyl, substituted or unsubstituted C₄-C₂₀ aryl, substituted orunsubstituted C₅-C₂₀ arylalkyl, substituted or unsubstituted 5- to15-membered heteroaryl, substituted or unsubstituted 5- to 15-memberedheteroaryl-C₁-C₂₀ alkyl, or substituted or unsubstituted acyl, and* is a binding position to the other moieties.

(3) The compound according to the embodiment (1) or (2), wherein A is amodifying group represented by the following formula (V), (VI), (VII) or(VIII):

whereina is an integer of 1 or larger,M³, M^(3′), M^(3″), M^(3′″) and M^(3″″) are each independently a bond ora polyethylene glycol group represented by formula (III):^(#)—(CH₂CH₂O)_(n)—**  (III)whereinn is an integer of 1 or larger,** is a binding position to R³, R^(3′) or CH, and# is a binding position to O,wherein when a plurality of M³, M^(3′), M^(3″), M^(3′″) or M^(3″″) arepresent, the plurality of M³, M^(3′), M^(3″), M^(3′″) or M^(3″″) are thesame as or different from each other, and at least one of M³, M^(3′),M^(3″), M^(3′″) and M^(3″″) is a polyethylene glycol group representedby formula (III),R¹, R^(1′), R^(1″) and R^(1′″) are each independently hydrogen,substituted or unsubstituted C₁-C₂₀ alkyl, substituted or unsubstitutedC₂-C₂₀ alkenyl, substituted or unsubstituted C₂-C₂₀ alkynyl, substitutedor unsubstituted C₃-C₂₀ cycloalkyl, substituted or unsubstituted C₄-C₂₀cycloalkenyl, substituted or unsubstituted C₄-C₂₀ cycloalkynyl,substituted or unsubstituted 3- to 6-membered heterocycloalkyl,substituted or unsubstituted C₇-C₂₀ cycloalkylalkyl, substituted orunsubstituted 3- to 6-membered heterocycloalkyl-C₁-C₂₀ alkyl,substituted or unsubstituted C₄-C₂₀ aryl, substituted or unsubstitutedC₅-C₂₀ arylalkyl, substituted or unsubstituted 5- to 15-memberedheteroaryl, substituted or unsubstituted 5- to 15-memberedheteroaryl-C₁-C₂₀ alkyl, or substituted or unsubstituted acyl,R² is a bond, substituted or unsubstituted C₁-C₂₀ alkylene, substitutedor unsubstituted C₂-C₂₀ alkenylene, substituted or unsubstituted C₂-C₂₀alkynylene, substituted or unsubstituted C₃-C₂₀ cycloalkylene,substituted or unsubstituted C₄-C₂₀ cycloalkenylene, substituted orunsubstituted C₄-C₂₀ cycloalkynylene, substituted or unsubstituted 3- to6-membered heterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, or substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene (the groupsoptionally comprise one or more heteroatoms, an amide group (—CO—NH—),an ester group (—CO—O—), or a urethane group (—O—CO—NH—)), an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—),R³, R^(3′) and R^(3″) are each independently a bond, substituted orunsubstituted C₁-C₂₀ alkylene, substituted or unsubstituted C₂-C₂₀alkenylene, substituted or unsubstituted C₂-C₂₀ alkynylene, substitutedor unsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstitutedC₄-C₂₀ cycloalkenylene, substituted or unsubstituted C₄-C₂₀cycloalkynylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, or substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene (the groupsoptionally comprise one or more heteroatoms, an amide group (—CO—NH—),an ester group (—CO—O—), or a urethane group (—O—CO—NH—)), an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—), wherein when a plurality of R³, R^(3′) or R^(3″) arepresent, the plurality of R³, R^(3′) or R^(3″) are the same as ordifferent from each other, and* is a binding position to the other moieties.

(4) The compound according to any of the embodiments (1) to (3), whereinthe polyethylene glycol group represented by formula (III) has aweight-average molecular weight ranging from 1 to 100 kDa in total.

(5) The compound according to any of the embodiments (1) to (4), whereinthe adrenomedullin or the modified form thereof with adrenomedullinactivity is a peptide selected from the group consisting of:

(i) a peptide consisting of an amino acid sequence of adrenomedullin,

(ii) a peptide that consists of an amino acid sequence of adrenomedullinand has a disulfide bond formed by two cysteine residues in the aminoacid sequence,

(iii) the peptide of (ii) wherein the disulfide bond of the peptide issubstituted with an ethylene group and the peptide has adrenomedullinactivity,

(iv) any peptide of (i) to (iii) wherein the peptide has the amino acidsequence comprising deletion, substitution, or addition of one tofifteen amino acid residues and has adrenomedullin activity,

(v) any peptide of (i) to (iv) wherein the peptide is amidated at theC-terminus thereof, and (vi) any peptide of (i) to (iv) wherein thepeptide has a glycine residue added to the C-terminus thereof.

(6) The compound according to the embodiment (5), wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of:

(i) a peptide consisting of an amino acid sequence of adrenomedullin,

(ii) a peptide that consists of an amino acid sequence of adrenomedullinand has a disulfide bond formed by two cysteine residues in the aminoacid sequence,

(v) the peptide of (i) or (ii) wherein the peptide is amidated at theC-terminus thereof, and (vi) the peptide of (i) or (ii) wherein thepeptide has a glycine residue added to the C-terminus thereof.

(7) The compound according to the embodiment (5), wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of:

(iv′) any peptide of (i) to (iii) wherein the peptide has deletion ofamino acid residues at positions 1 to 15, positions 1 to 10, orpositions 1 to 5 from the N-terminus thereof and has adrenomedullinactivity,

(v) the peptide of (iv′) wherein the peptide is amidated at theC-terminus thereof, and

(vi) the peptide of (iv′) wherein the peptide has a glycine residueadded to the C-terminus thereof.

(8) The compound according to any of the embodiments (1) to (5), whereinthe adrenomedullin or the modified form thereof is a peptide selectedfrom the group consisting of:

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, ora peptide consisting of the amino acid sequence of SEQ ID NO: 1 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, ora peptide consisting of the amino acid sequence of SEQ ID NO: 3 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, ora peptide consisting of the amino acid sequence of SEQ ID NO: 5 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, ora peptide consisting of the amino acid sequence of SEQ ID NO: 9 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, ora peptide consisting of the amino acid sequence of SEQ ID NO: 11 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(g) any peptide of (a) to (f) wherein the disulfide bond of the peptideis substituted with an ethylene group and the peptide has adrenomedullinactivity;

(h) any peptide of (a) to (g) wherein the peptide has the amino acidsequence comprising deletion, substitution, or addition of one tofifteen amino acids and has adrenomedullin activity;

(i) any peptide of (a) to (h) wherein the peptide is amidated at theC-terminus thereof; and

(j) any peptide of (a) to (h) wherein the peptide has a glycine residueadded to the C-terminus thereof.

(9) The compound according to the embodiment (8), wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of:

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, ora peptide consisting of the amino acid sequence of SEQ ID NO: 1 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, ora peptide consisting of the amino acid sequence of SEQ ID NO: 3 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, ora peptide consisting of the amino acid sequence of SEQ ID NO: 5 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, ora peptide consisting of the amino acid sequence of SEQ ID NO: 9 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, ora peptide consisting of the amino acid sequence of SEQ ID NO: 11 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(i) any peptide of (a) to (f) wherein the peptide is amidated at theC-terminus thereof; and

(j) any peptide of (a) to (f) wherein the peptide has a glycine residueadded to the C-terminus thereof.

(10) The compound according to the embodiment (8), wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of:

(h′) any peptide of (a) to (d) wherein the peptide has deletion of aminoacid residues at positions 1 to 15, positions 1 to 10, or positions 1 to5 from the N-terminus thereof and has adrenomedullin activity, or thepeptide of (e) or (f) wherein the peptide has deletion of amino acidresidues at positions 1 to 13, positions 1 to 8, or positions 1 to 5from the N-terminus thereof and has adrenomedullin activity;(i) the peptide of (h′) wherein the peptide is amidated at theC-terminus thereof; and(j) the peptide of (h′) wherein the peptide has a glycine residue addedto the C-terminus thereof.

(11) A method for producing the compound according to any of theembodiments (1) to (10) or a salt thereof, or a hydrate thereof,comprising a linking step of reacting a precursor of peptide moiety Bderived from adrenomedullin or a modified form thereof with a precursoraldehyde of modifying group A comprising one or more polyethylene glycolgroups in the presence of a reducing agent to form the compoundrepresented by formula (I), wherein the precursor aldehyde isrepresented by formula (I-1):A-CHO  (I-1).

(12) A compound represented by formula (X):A′-CO—B  (X)whereinA′ is a modifying group comprising one or more polyethylene glycolgroups, andB is a peptide moiety derived from adrenomedullin or a modified formthereof with adrenomedullin activity,wherein the peptide moiety B is linked to the other moieties through acovalent bond of the nitrogen atom of the N-terminal α-amino group ofthe peptide moiety B to the carbon atom of the carbonyl group,A′ is a modifying group represented by the following formula (XI), (XI′)or (XII):

whereina is an integer of 1 or larger,M¹ is a polyethylene glycol group represented by formula (III):^(#)—(CH₂CH₂O)_(n)—**  (III)whereinn is an integer of 1 or larger,** is a binding position to *, and# is a binding position to O,M³, M^(3′) and M^(3″) are each independently a bond or a polyethyleneglycol group represented by formula (III):^(#)—(CH₂CH₂O)_(n)—**  (III)whereinn is an integer of 1 or larger,** is a binding position to R³, R^(3′) or CH, and# is a binding position to O,wherein when a plurality of M³, M^(3′) or M^(3″) are present, theplurality of M³, M^(3′) or M^(3″) are the same as or different from eachother, and at least one of M³, M^(3′) and M^(3″) is a polyethyleneglycol group represented by formula (III),R¹ and R^(1′) are each independently hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted C₂-C₂₀ alkenyl,substituted or unsubstituted C₂-C₂₀ alkynyl, substituted orunsubstituted C₃-C₂₀ cycloalkyl, substituted or unsubstituted C₄-C₂₀cycloalkenyl, substituted or unsubstituted C₄-C₂₀ cycloalkynyl,substituted or unsubstituted 3- to 6-membered heterocycloalkyl,substituted or unsubstituted C₇-C₂₀ cycloalkylalkyl, substituted orunsubstituted 3- to 6-membered heterocycloalkyl-C₁-C₂₀ alkyl,substituted or unsubstituted C₄-C₂₀ aryl, substituted or unsubstitutedC₅-C₂₀ arylalkyl, substituted or unsubstituted 5- to 15-memberedheteroaryl, substituted or unsubstituted 5- to 15-memberedheteroaryl-C₁-C₂₀ alkyl, or substituted or unsubstituted acyl,R² is a bond, substituted or unsubstituted C₁-C₂₀ alkylene, substitutedor unsubstituted C₂-C₂₀ alkenylene, substituted or unsubstituted C₂-C₂₀alkynylene, substituted or unsubstituted C₃-C₂₀ cycloalkylene,substituted or unsubstituted C₄-C₂₀ cycloalkenylene, substituted orunsubstituted C₄-C₂₀ cycloalkynylene, substituted or unsubstituted 3- to6-membered heterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, or substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene (the groupsoptionally comprise one or more heteroatoms, an amide group (—CO—NH—),an ester group (—CO—O—), or a urethane group (—O—CO—NH—)), an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—),R³, R^(3′) and R^(3″) are each independently a bond, substituted orunsubstituted C₁-C₂₀ alkylene, substituted or unsubstituted C₂-C₂₀alkenylene, substituted or unsubstituted C₂-C₂₀ alkynylene, substitutedor unsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstitutedC₄-C₂₀ cycloalkenylene, substituted or unsubstituted C₄-C₂₀cycloalkynylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, or substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene (the groupsoptionally comprise one or more heteroatoms, an amide group (—CO—NH—),an ester group (—CO—O—), or a urethane group (—O—CO—NH—)), an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—), wherein when a plurality of R³, R^(3′) or R^(3″) arepresent, the plurality of R³, R^(3′) or R^(3″) are the same as ordifferent from each other, and* is a binding position to the other moieties,or a salt thereof, or a hydrate thereof.

(13) A medicament comprising the compound according to any of theembodiments (1) to (10) and (12) or a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable hydrate thereof as an activeingredient.

(14) The medicament according to the embodiment (13) for use in theprevention or treatment of a cardiovascular disease, an inflammatorydisease, or a peripheral vascular disease.

(15) An agent for preventing or treating a cardiovascular disease, aninflammatory disease, or a peripheral vascular disease, wherein theagent comprises the compound according to any of the embodiments (1) to(10) and (12) or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable hydrate thereof as an active ingredient.

(16) A pharmaceutical composition comprising the compound according toany of the embodiments (1) to (10) and (12) or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable hydratethereof and one or more pharmaceutically acceptable carriers.

(17) The pharmaceutical composition according to the embodiment (16) foruse in the prevention or treatment of a cardiovascular disease, aninflammatory disease, or a peripheral vascular disease.

(18) A method for preventing or treating a condition, disease, and/ordisorder, comprising administering to a subject in need of prevention ortreatment of the condition, disease, and/or disorder an effective amountof the compound according to any of the embodiments (1) to (10) and (12)or a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable hydrate thereof.

(19) The method according to the embodiment (18) wherein the condition,disease, and/or disorder is a cardiovascular disease, an inflammatorydisease, or a peripheral vascular disease.

(20) The compound according to any of the embodiments (1) to (10) and(12) or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable hydrate thereof for use in the prevention ortreatment of a condition, disease, and/or disorder.

(21) The compound according to the embodiment (20) wherein thecondition, disease, and/or disorder is a cardiovascular disease, aninflammatory disease, or a peripheral vascular disease.

(22) Use of the compound according to any of the embodiments (1) to (10)and (12) or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable hydrate thereof for manufacturing amedicament for the prevention or treatment of a condition, disease,and/or disorder.

(23) The use according to the embodiment (22) wherein the condition,disease, and/or disorder is a cardiovascular disease, an inflammatorydisease, or a peripheral vascular disease.

Advantageous Effects of Invention

The invention can provide novel adrenomedullin derivatives sustainablefor a long period which are capable of substantially suppressingunwanted side effects while maintaining pharmacological effects ofadrenomedullin.

The present specification includes contents described in thespecification and/or drawings of Japanese patent application No.2015-184685 to which the present application claims priority.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows reverse-phase HPLC (RP-HPLC) chromatograms of cleavedpeptides. A: RP-HPLC chromatogram of a cleaved peptide derived from h.AM(1-52) peptide; and B: RP-HPLC chromatogram of a cleaved peptide derivedfrom compound (2).

FIG. 2 shows results of separating compounds (3), (4), (5), (6), (7),(8), (9), (10), (11) and (12) by SDS-PAGE using a polyacrylamide gelwith a concentration gradient from 10% to 20%. In the diagram, lane 0depicts a molecular weight standard, lane 1 depicts the compound (3),lane 2 depicts the compound (4), lane 3 depicts the compound (5), lane 4depicts the compound (6), lane 5 depicts the compound (7), lane 6depicts the compound (8), lane 7 depicts the compound (9), lane 8depicts the compound (10), lane 9 depicts the compound (11), and lane 10depicts the compound (12).

FIG. 3 shows results of separating compounds (1), (2), (13), (14), (15),(16) and (17) by SDS-PAGE using a polyacrylamide gel with aconcentration gradient from 10% to 20%. In the diagram, lane 0 depicts amolecular weight standard, lane 1 depicts the compound (1), lane 2depicts the compound (2), lane 3 depicts the compound (13), lane 4depicts the compound (14), lane 5 depicts the compound (15), lane 6depicts the compound (16), and lane 7 depicts the compound (17).

FIG. 4 shows results of separating compounds (25), (26), (27), (28),(29), (30), (31), (32), (33), (34), (35), (36) and (37) by SDS-PAGEusing a polyacrylamide gel with a concentration gradient from 10% to20%. In the diagram, lane 0 depicts a molecular weight standard, lane 1depicts the compound (25), lane 2 depicts the compound (26), lane 3depicts the compound (27), lane 4 depicts the compound (28), lane 5depicts the compound (29), lane 6 depicts the compound (30), lane 7depicts the compound (31), lane 8 depicts the compound (32), lane 9depicts the compound (33), lane 10 depicts the compound (34), lane 11depicts the compound (35), lane 12 depicts the compound (36), and lane13 depicts the compound (37).

FIG. 5 shows results of separating compounds (18), (19), (20), (21),(22), (23) and (24) by SDS-PAGE using a polyacrylamide gel with aconcentration gradient from 10% to 20%. In the diagram, lanes 0 and 1depict a molecular weight standard, lane 2 depicts the compound (18),lane 3 depicts the compound (19), lane 4 depicts the compound (20), lane5 depicts the compound (21), lane 6 depicts the compound (22), lane 7depicts the compound (23), and lane 8 depicts the compound (24).

FIG. 6 shows the relationship between the time elapsed from the start ofadministration of compound (2), compound (4), compound (8) or h.AM(1-52) and the average blood pressure. A: results about the compound(2), the compound (4) and h.AM (1-52); and B: results about the compound(8) and h.AM (1-52).

FIG. 7 shows the relationship between the time elapsed from the start ofadministration of compound (8) and AM concentration in blood plasma.

FIG. 8 shows the relationship between the time elapsed from the start ofadministration of compound (6) or h.AM (1-52) and AM concentration inblood plasma.

FIG. 9 shows the blood pressure values of spontaneously hypertensiverats 2 days before and 9 days after administration of compound (8) orphysiological saline.

FIG. 10 shows the values of change in the blood pressure obtained 4 daysand 9 days after administration of compound (37) or physiological salinewith respect to the average systolic blood pressure on the day beforeadministration.

FIG. 11 shows the relationship between the time elapsed from dextransodium sulfate (DSS)-induced colitis model preparation and the totalvalues of scores in the compound (8) administration group and thecontrol group.

FIG. 12 shows the relationship between the time elapsed from2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis modelpreparation and body weights in the compound (8) administration groupand the control group. a: day on which compound (8) or physiologicalsaline was subcutaneously administered and fasting was started; and b:day on which TNBS was administered.

FIG. 13 shows the weights of the large intestines in the compound (8)administration group and the control group.

FIG. 14 shows the intestinal tract lengths of the large intestines inthe compound (8) administration group and the control group.

FIG. 15 shows right ventricle weight/left ventricle weight ratios in thecompound (8) administration group and the control group.

FIG. 16 shows the relationship between the time elapsed from wound modelpreparation and wound areas in the compound (8) administration group andthe control group.

FIG. 17 shows the relationship between the time elapsed from vascularocclusion model preparation and escape latency in the hidden platformtest in the compound (8) administration group and the control group.

FIG. 18 shows stay rates in the probe test in the compound (8)administration group and the control group of vascular occlusion modelrats.

FIG. 19 shows the relationship between the time elapsed fromadministration of compound (8) or a vehicle and paw volumes exhibitedafter adjuvant administration in the compound (8) administration groupand the control group.

FIG. 20 shows the relationship between the time elapsed fromadministration of compound (8) or a vehicle and swelling rates exhibitedafter adjuvant administration in the compound (8) administration groupand the control group.

FIG. 21 shows the relationship between the time elapsed fromadministration of compound (8) or a vehicle and inflammation scoresexhibited after adjuvant administration in the compound (8)administration group and the control group.

DESCRIPTION OF EMBODIMENTS

<1. Adrenomedullin Derivative>

An aspect of the invention relates to a compound represented by formula(I):A-CH₂—B  (I)or a salt thereof, or a hydrate thereof. In the present specification,the compound represented by formula (I) may be described as“adrenomedullin derivative”.

In the invention, adrenomedullin (AM) may not only be a peptide derivedfrom human and isolated and identified from human pheochromocytoma (SEQID NO: 1, Non Patent Literature 1), but also be a peptide derived fromother non-human mammals (such as warm-blooded animals), which is anortholog, including, for example, pig (SEQ ID NO: 3), dog (SEQ ID NO:5), cattle (SEQ ID NO: 7), rat (SEQ ID NO: 9), or mouse (SEQ ID NO: 11).In a living body, each of these peptides has a disulfide bond formed bytwo cysteine residues in the amino acid sequence and is amidated at theC-terminus thereof. In the present specification, the peptide having adisulfide bond and C-terminal amide group may be described as “naturaladrenomedullin” or simply “adrenomedullin”. The invention can be appliedto any of the peptides described above.

In the present specification, “C-terminal amidation” means an aspect ofpost-translational modification of a peptide in a living body, andspecifically means a reaction in which the main chain carboxyl group ofC-terminal amino acid residue of the peptide is converted into an amidegroup. In the present specification, “formation of a disulfide bondbetween cysteine residues” or “disulfide bond formation by cysteineresidues” means an aspect of post-translational modification of thepeptide in a living body, and specifically means a reaction in which twocysteine residues in the amino acid sequence of the peptide form adisulfide bond (—S—S—). Many bioactive peptides produced in a livingbody are initially biosynthesized as a precursor protein with largermolecular weight. The precursor protein is subject to post-translationalmodifications, such as C-terminal amidation and/or disulfide bondformation by cysteine residues, during the process of intracellulartransport to give a mature bioactive peptide. The C-terminal amidationtypically proceeds by a C-terminal amidating enzyme that acts on theprecursor protein. For a bioactive peptide having a C-terminal amidegroup, the precursor protein has a Gly residue bound to the C-terminalcarboxyl group to be amidated and the Gly residue is converted into theC-terminal amide group by the C-terminal amidating enzyme. TheC-terminal propeptide in the precursor protein has a repeat sequencecomprising a combination of basic amino acid residues, such as Lys-Argor Arg-Arg (Mizuno, Journal of Japanese Biochemical Society, 61(12):1435-1461 (1989)). Disulfide bond formation by cysteine residues canproceed under oxidative conditions. Disulfide bond formation by cysteineresidues in a living body typically proceeds by a protein disulfideisomerase that acts on the precursor protein.

Adrenomedullin, a known bioactive substance, is a peptide. This maycause a medicament comprising adrenomedullin as an active ingredient toact effectively in living bodies in subjects (such as human patients)only for a very short time. Accordingly, attempts have been made toprolong half-life in a living body and improve pharmacokinetics by meansof forms of adrenomedullin derivatives in which other groups such aspolyethylene glycol (PEG) are linked to adrenomedullin (PatentLiteratures 4 to 6 and Non Patent Literature 10). However, in the caseof linking a relatively large group such as a PEG group to a relativelysmall peptide such as adrenomedullin, various properties of theresulting adrenomedullin derivative may vary largely depending on themolecular weight of the PEG group. When adrenomedullin and other groupsare linked through a bond, such as an amide bond or an ester bond, whichmay be cleaved by a biological reaction, the bond may be cleaved in arelatively short time after administration. In the case of linking othergroups to the side chain of an amino acid residue of adrenomedullin, theconformation of the adrenomedullin moiety may be changed to reduce theaffinity for an AM receptor recognizing adrenomedullin. In such a case,pharmacological effects as adrenomedullin of the resultingadrenomedullin derivative may be reduced.

Adrenomedullin has a strong vasodilatory effect. This strongvasodilatory effect may cause unwanted side effects (such as excessivedecreased blood pressure, tachycardia associated with increased reflexsympathetic nerve activity, and/or increased activity of renin) when atherapeutically effective amount of adrenomedullin or a derivativethereof is administered in a single dose. The occurrence of such sideeffects may become a problem when adrenomedullin or a derivative thereofis used, particularly, in the expectation that pharmacological effectsother than a vasodilatory effect are exerted. To avoid generating theproblems described above, a medicament comprising adrenomedullin or aderivative thereof as an active ingredient is required to beadministered to subjects via continuous intravenous infusion. Such amode of administration may force subjects to bear an undue burden.

The present inventors have found that linking of the N-terminal α-aminogroup of adrenomedullin to a PEG group with a specific molecular weightvia a methylene group or a urethane group can prolong blood half-life ofthe resulting adrenomedullin derivative as compared to adrenomedullinwhile retaining bioactivity of adrenomedullin. The present inventorshave further found that novel adrenomedullin derivatives having theproperties described above are capable of substantially suppressingunwanted side effects such as excessive decreased blood pressure. Thus,the compound of the invention represented by formula (I) can be appliedto a condition, disease, and/or disorder that can be prevented ortreated with adrenomedullin to sustainably prevent or treat thecondition, disease, and/or disorder while substantially suppressingunwanted side effects.

In the formula (I), B is required to be a peptide moiety derived fromadrenomedullin or a modified form thereof with adrenomedullin activity.In the invention, “peptide moiety derived from adrenomedullin or amodified form thereof with adrenomedullin activity” means a monovalentfree group with a structure derived from adrenomedullin or the modifiedform thereof with adrenomedullin activity by removal of one hydrogenatom (commonly, one hydrogen atom of an amino group, typically onehydrogen atom of the N-terminal (α-amino group). In the invention, “amodified form of adrenomedullin” means a peptide chemically modifiedfrom natural adrenomedullin as described above. In the invention,“adrenomedullin activity” means bioactivity that adrenomedullin has. Theadrenomedullin activity can include the following:

(1) Cardiovascular: a vasodilatory effect, an effect of lowering bloodpressure, an effect of suppressing increase in blood pressure, an effectof increasing cardiac output or improving cardiac insufficiency, aneffect of improving pulmonary hypertension, an angiogenic effect, alymphangiogenic effect, an effect of improving vascular endothelialfunction, an antiarteriosclerotic effect, a myocardial protective effect(such as a myocardial protective effect in ischemic reperfusion disorderor inflammation), an effect of preventing postmyocardial remodeling, aneffect of suppressing cardiac hypertrophy, and an effect of suppressingan angiotensin-converting enzyme.

(2) Kidney and water and electrolyte system: a diuretic effect, anatriuretic effect, an effect of suppressing antidiuretic hormone, analdosterone-reducing effect, a renoprotective effect (such as arenoprotective effect in high blood pressure or ischemic reperfusiondisorder), an effect of suppressing drinking behavior, and an effect ofsuppressing salt requirement.

(3) Brain and nervous system: an effect of neuroprotection andpreventing encephalopathy, an anti-inflammatory effect, an effect ofsuppressing apoptosis (such as an effect of suppressing apoptosis inischemic reperfusion disorder or inflammation), an effect of maintainingautoregulatory capacity, an effect of suppressing oxidative stress, aneffect of improving dementia, and a sympathoinhibitory effect.

(4) Urogenital: an effect of improving erection, an effect of improvingblood flow, and an implantation-promoting effect.

(5) Gastrointestinal system: an antiulcer effect, a tissue repaireffect, an effect of neogenesis of mucous membrane, an effect ofimproving blood flow, an anti-inflammatory effect, and an effect ofimproving liver function.

(6) Orthopedics: an effect of stimulating osteoblast and an effect ofimproving arthritis.

(7) Endocrine metabolic system: an adipocyte-differentiating effect, aneffect of regulating lipolysis, an effect of improving insulinsensitivity, an effect of controlling insulin secretion, an effect ofsuppressing antidiuretic hormone secretion, and an effect of suppressingaldosterone secretion.

(8) Other: an effect of improving circulation, an anti-inflammatoryeffect, an effect of modulating cytokine, an organ protective effect, aneffect of suppressing oxidative stress, an effect of repairing tissue(such as an anti-decubitus effect), an effect of improving septic shock,an effect of suppressing multiple organ failure, an effect ofsuppressing auto-immune disease, an antimicrobial effect, a hair growtheffect, and a pilatory effect.

The blood pressure-lowering effect is preferably a vasodilatoryhypotensive effect. The anti-inflammatory effect in the gastrointestinalsystem is preferably an effect of preventing or treating inflammatorybowel diseases including a steroid-resistant or steroid-dependentinflammatory bowel disease (such as ulcerative colitis, Crohn's disease,or intestinal tract Behcet's disease). The adrenomedullin activity willbe exerted via increased concentration of intracellular cAMP. Thus, theincreased concentration of intracellular cAMP can be considered as anindex of adrenomedullin activity. The peptide moiety B derived fromadrenomedullin or a modified form thereof having the bioactivity asdescribed above enables the compound of the invention represented byformula (I) to exert bioactivity substantially approximately equivalentto that of natural adrenomedullin (i.e., adrenomedullin activity).

The adrenomedullin or a modified form thereof with adrenomedullinactivity is preferably a peptide selected from the group consisting of:

(i) a peptide consisting of an amino acid sequence of adrenomedullin,

(ii) a peptide that consists of an amino acid sequence of adrenomedullinand has a disulfide bond formed by two cysteine residues in the aminoacid sequence,

(iii) the peptide of (ii) wherein the disulfide bond of the peptide issubstituted with an ethylene group and the peptide has adrenomedullinactivity,

(iv) any peptide of (i) to (iii) wherein the peptide has the amino acidsequence comprising deletion, substitution, or addition of one tofifteen amino acids and has adrenomedullin activity,

(v) any peptide of (i) to (iv) wherein the peptide is amidated at theC-terminus thereof, and

(vi) any peptide of (i) to (iv) wherein the peptide has a glycineresidue added to the C-terminus thereof.

In one embodiment, the adrenomedullin or a modified form thereof withadrenomedullin activity is more preferably a peptide selected from thegroup consisting of:

(i) a peptide consisting of an amino acid sequence of adrenomedullin,

(ii) a peptide that consists of an amino acid sequence of adrenomedullinand has a disulfide bond formed by two cysteine residues in the aminoacid sequence,

(v) the peptide of (i) or (ii) wherein the peptide is amidated at theC-terminus thereof, and

(vi) the peptide of (i) or (ii) wherein the peptide has a glycineresidue added to the C-terminus thereof.

In another embodiment, the adrenomedullin or a modified form thereofwith adrenomedullin activity is more preferably a peptide selected fromthe group consisting of:

(iv′) any peptide of (i) to (iii) wherein the peptide has deletion ofamino acid residues at positions 1 to 15, positions 1 to 10, orpositions 1 to 5 from the N-terminus thereof and has adrenomedullinactivity,

(v) the peptide of (iv′) wherein the peptide is amidated at theC-terminus thereof, and

(vi) the peptide of (iv′) wherein the peptide has a glycine residueadded to the C-terminus thereof.

In the peptides of (i) to (vi) and (iv′), a peptide involved in (v),which consists of the amino acid sequence of adrenomedullin, is amidatedat the C-terminus thereof, and has a disulfide bond formed by twocysteine residues in the amino acid sequence, represents a maturenatural adrenomedullin. A peptide of (i) consisting of an amino acidsequence of adrenomedullin represents a form (i.e., an immature form) ofnatural adrenomedullin prior to post-translational modificationincluding C-terminal amidation and disulfide bond formation by cysteineresidues. Other peptides except peptides described above in the peptidesof (i) to (vi) and (iv′) represent modified forms of adrenomedullin.

The peptide of (ii) can be formed by oxidizing thiol groups of twocysteine residues in the peptide of (i) with air or with a suitableoxidizing agent to form a disulfide bond. The peptide of (ii) can beused to establish the conformation of the peptide moiety B similar tothat of natural adrenomedullin. This similar conformation can leadadrenomedullin activity of a compound represented by formula (I) to anactivity substantially approximately equivalent to that of naturaladrenomedullin.

The peptide of (iii) can be formed by converting the disulfide bond inthe peptide of (ii) into an ethylene group. The substitution of thedisulfide bond to an ethylene group can be accomplished by any methodwell known in the art (O. Keller et al., Helv. Chim. Acta, 1974, Volume57, p. 1253). The peptide of (iii) can be used to stabilize theconformation of peptide moiety B. The stabilized conformation allows acompound represented by formula (I) to sustainably exert adrenomedullinactivity in a living body.

In the peptide of (iv), the number of amino acid residues deleted,substituted, or added preferably ranges from 1 to 15, more preferablyfrom 1 to 10, further preferably from 1 to 8, especially preferably from1 to 5, and most preferably from 1 to 3. A suitable peptide of (iv) isany peptide of (i) to (iii) wherein the peptide has deletion of aminoacid residues at positions 1 to 15, positions 1 to 12, positions 1 to10, positions 1 to 8, positions 1 to 5, or positions 1 to 3 from theN-terminus thereof and has adrenomedullin activity. A more suitablepeptide of (iv) is any peptide of (i) to (iii) wherein the peptide hasdeletion of amino acid residues at positions 1 to 15, positions 1 to 10,or positions 1 to 5 from the N-terminus thereof and has adrenomedullinactivity (peptide of (iv′)). The suitable peptide may have furtherdeletion, substitution, or addition of one or more (such as 1 to 5, 1 to3, or 1 or 2) amino acid residues.

The peptide of (iv) or (iv′) can be used to achieve adrenomedullinactivity of a compound represented by formula (I) substantiallyapproximately equivalent to that of natural adrenomedullin. Also, thepeptide of (iv) or (iv′) can be used to sustainably exert adrenomedullinactivity of a compound represented by formula (I) in a living body.

The peptide of (vi) or (iv′) can be converted to the peptide of (v) by aC-terminal amidating enzyme which can convert a glycine residue at theC-terminus of the peptide of (vi) or (iv′) into an amide group.Therefore, the peptide of (vi) or (iv′) can be administered to a subjectto form the peptide amidated at the C-terminus thereof in the livingbody of the subject after a certain period of time. Thus, a compoundrepresented by formula (I) can sustainably exert adrenomedullin activityin a living body.

The adrenomedullin or a modified form thereof is more preferably apeptide selected from the group consisting of:

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, ora peptide consisting of the amino acid sequence of SEQ ID NO: 1 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(b) a peptide consisting of the amino acid sequence of the SEQ ID NO: 3,or a peptide consisting of the amino acid sequence of the SEQ ID NO: 3and having a disulfide bond formed by the cysteine residues at positions16 and 21;

(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, ora peptide consisting of the amino acid sequence of SEQ ID NO: 5 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, ora peptide consisting of the amino acid sequence of SEQ ID NO: 9 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, ora peptide consisting of the amino acid sequence of SEQ ID NO: 11 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(g) any peptide of (a) to (f) wherein the disulfide bond of the peptideis substituted with an ethylene group and the peptide has adrenomedullinactivity;

(h) any peptide of (a) to (g) wherein the peptide has the amino acidsequence comprising deletion, substitution, or addition of one tofifteen amino acids and has adrenomedullin activity;

(i) any peptide of (a) to (h) wherein the peptide is amidated at theC-terminus thereof; and

(j) any peptide of (a) to (h) wherein the peptide has a glycine residueadded to the C-terminus thereof.

In one embodiment, the adrenomedullin or a modified form thereof isfurther preferably a peptide selected from the group consisting of:

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, ora peptide consisting of the amino acid sequence of SEQ ID NO: 1 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, ora peptide consisting of the amino acid sequence of SEQ ID NO: 3 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, ora peptide consisting of the amino acid sequence of SEQ ID NO: 5 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, ora peptide consisting of the amino acid sequence of SEQ ID NO: 9 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, ora peptide consisting of the amino acid sequence of SEQ ID NO: 11 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(i) any peptide of (a) to (f) wherein the peptide is amidated at theC-terminus thereof; and (j) any peptide of (a) to (f) wherein thepeptide has a glycine residue added to the C-terminus thereof.

In another embodiment, the adrenomedullin or a modified form thereof isfurther preferably a peptide selected from the group consisting of:

(h′) any peptide of (a) to (d) wherein the peptide has deletion of aminoacid residues at positions 1 to 15, positions 1 to 10, or positions 1 to5 from the N-terminus thereof and has adrenomedullin activity, or thepeptide of (e) or (f) wherein the peptide has deletion of amino acidresidues at positions 1 to 13, positions 1 to 8, or positions 1 to 5from the N-terminus thereof and has adrenomedullin activity;(i) the peptide of (h′) wherein the peptide is amidated at theC-terminus thereof; and(j) the peptide of (h′) wherein the peptide has a glycine residue addedto the C-terminus thereof.

In the peptide of (h), the number of amino acid residues deleted,substituted, or added preferably ranges from 1 to 12, more preferablyfrom 1 to 10, further preferably from 1 to 8, especially preferably from1 to 5, and most preferably from 1 to 3. A suitable peptide of (h) isany peptide of (a) to (g) wherein the peptide has deletion of amino acidresidues at positions 1 to 15, positions 1 to 12, positions 1 to 10,positions 1 to 8, positions 1 to 5, or positions 1 to 3 from theN-terminus thereof and has adrenomedullin activity. A more suitablepeptide of (h) is any peptide of (a) to (d) wherein the peptide hasdeletion of amino acid residues at positions 1 to 15, positions 1 to 10,or positions 1 to 5 from the N-terminus thereof and has adrenomedullinactivity, or the peptide of (e) or (f) wherein the peptide has deletionof amino acid residues at positions 1 to 13, positions 1 to 8, orpositions 1 to 5 from the N-terminus thereof and has adrenomedullinactivity (peptide of (h′)). The suitable peptide may have furtherdeletion, substitution, or addition of one or more (such as 1 to 5, 1 to3, or 1 or 2) amino acids. The peptide of (h) or (h′) can be used toachieve adrenomedullin activity of a compound represented by formula (I)substantially approximately equivalent to that of naturaladrenomedullin. Also, the peptide of (h) or (h′) can be used tosustainably exert adrenomedullin activity of a compound represented byformula (I) in a living body.

In the formula (I), A is required to be a modifying group comprising oneor more PEG groups. An aspect of the modifying group A comprising one ormore PEG groups is not particularly limited. For example, one or morePEG groups may be positioned at the terminal site of the modifying groupA or may be positioned in the inside of the modifying group A. Also, themodifying group A may be various groups known in the art as PEGgroup-containing linear or branched groups. Known groups that can beused as the modifying group A can include, but are not limited to, forexample, groups disclosed in WO1995/11924, WO2006/084089, WO98/41562,WO2005/079838, WO2002/060978, WO2001/048052, WO1998/055500,WO1996/021469, WO2003/040211, and JP Patent Publication (Kokai) No.04-108827 A (1992). By use of a group comprising one or more PEG groupsas the modifying group A, a compound represented by formula (I) cansustainably exert adrenomedullin activity in a living body.

A is preferably a modifying group represented by the following formula(II):

In the formula (II),

a is an integer of 1 or larger,

m is an integer of 1 or larger,

L¹ is a m+1-valent linear or branched linking group, wherein when aplurality of L¹ are present, the plurality of L¹ are the same as ordifferent from each other,

L² and L^(2′) are each independently a bond or a divalent linking group,wherein when a plurality of L^(2′) are present, the plurality of L^(2′)are the same as or different from each other,

M¹ is a PEG group, wherein when a plurality of M¹ are present, theplurality of M¹ are the same as or different from each other,

M² is a bond or a PEG group, wherein when a plurality of M² are present,the plurality of M² are the same as or different from each other,

R¹ is hydrogen or a monovalent group, and

* is a binding position to the other moieties.

m is the number of branches of the linking group L¹. For example, when mis 1, L¹ is a divalent linking group and is a group non-branched towardthe terminal directions, i.e., a linear group. When m is 2 or larger, L¹is a trivalent or higher linking group and is a group having two or morebranches toward the terminal directions. m is typically an integer of 1or larger and 5 or smaller and preferably ranges from 1 to 5, morepreferably from 1 to 4, and further preferably from 1 to 3. When thenumber m of branches of the linking group L¹ falls within the rangesdescribed above, the modifying group A comprising PEG groups can have alinear or branched structure.

a is the number of repeats of a unit containing the PEG groups M¹ and M²and the linking groups L¹ and L^(2′). For example, when a is 1, the unithas no repeat structure. When a is 2 or larger and m is 1, the unit hasa linear repeat structure. When a is 2 or larger and m is 2 or larger,the unit has a dendritically branched repeat structure. a is typicallyan integer of 1 or larger and 5 or smaller and preferably ranges from 1to 5 and more preferably from 1 to 2. The number a of repeats of theunit containing the PEG groups M¹ and M² and the linking groups L¹ andL^(2′) falls within the ranges described above, the modifying group Acomprising PEG groups can have a linear or branched structure.

The PEG group represented by M¹ or M² is typically a group representedby formula (III):^(#)—(CH₂CH₂O)_(n)—**  (III)In the formula (III), ** is a binding position to L¹, and # is a bindingposition to O or L^(2′). The weight-average molecular weight of the PEGgroup represented by formula (III) is typically 1 kDa or larger,preferably 5 kDa or larger, more preferably 10 kDa or larger, andfurther preferably 20 kDa or larger, and is typically 2000 kDa orsmaller, preferably 1000 kDa or smaller, more preferably 100 kDa orsmaller, further preferably 80 kDa or smaller, and especially preferably60 kDa or smaller, in total in the modifying group A. The PEG grouprepresented by formula (III) typically has a weight-average molecularweight ranging from 1 to 2000 kDa, for example, from 1 to 1000 kDa, andpreferably has a weight-average molecular weight ranging from 1 to 100kDa, more preferably from 5 to 80 kDa, further preferably from 10 to 60kDa, and especially from 20 to 60 kDa, in total in the modifying groupA. When the total weight-average molecular weight of the PEG grouprepresented by formula (III) in the modifying group A falls within theranges described above, a compound represented by formula (I) can haveadrenomedullin activity substantially approximately equivalent to thatof natural adrenomedullin. Furthermore, the compound represented byformula (I) can sustainably exert adrenomedullin activity in a livingbody while substantially suppressing unwanted side effects.

In the formula (III), n is the number of repeats of an ethylene oxideunit defined on the basis of the weight-average molecular weight. ndefined on the basis of the preferred ranges of the weight-averagemolecular weight is typically an integer of approximately 20 or larger,preferably approximately 110 or larger, more preferably approximately230 or larger, and further preferably approximately 460 or larger, andis typically an integer of approximately 45000 or smaller, preferablyapproximately 22000 or smaller, more preferably approximately 2200 orsmaller, further preferably approximately 1820 or smaller, andespecially preferably approximately 1360 or smaller. n defined on thebasis of the preferred ranges of the weight-average molecular weighttypically ranges from approximately 20 to 45000, for example, fromapproximately 20 to 22000, and preferably ranges from approximately 1 to2200, more preferably from approximately 110 to 1820, further preferablyfrom approximately 230 to 1360, and especially from approximately 460 to1360. When the number n of repeats falls within the ranges describedabove, the total weight-average molecular weight of PEG groups containedin the modifying group represented by formula (II) falls within theranges described above. Therefore, when the number n of repeats fallswithin the ranges described above, a compound represented by formula (I)can have adrenomedullin activity substantially approximately equivalentto that of natural adrenomedullin. Furthermore, the compound representedby formula (I) can sustainably exert adrenomedullin activity in a livingbody while substantially suppressing unwanted side effects.

R¹ is preferably hydrogen, substituted or unsubstituted C₁-C₂₀ alkyl,substituted or unsubstituted C₂-C₂₀ alkenyl, substituted orunsubstituted C₂-C₂₀ alkynyl, substituted or unsubstituted C₃-C₂₀cycloalkyl, substituted or unsubstituted C₄-C₂₀ cycloalkenyl,substituted or unsubstituted C₄-C₂₀ cycloalkynyl, substituted orunsubstituted 3- to 6-membered heterocycloalkyl, substituted orunsubstituted C₇-C₂₀ cycloalkylalkyl, substituted or unsubstituted 3- to6-membered heterocycloalkyl-C₁-C₂₀ alkyl, substituted or unsubstitutedC₄-C₂₀ aryl, substituted or unsubstituted C₅-C₂₀ arylalkyl, substitutedor unsubstituted 5- to 15-membered heteroaryl, substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkyl, or substitutedor unsubstituted acyl, more preferably hydrogen, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted C₂-C₂₀ alkenyl,or substituted or unsubstituted C₂-C₂₀ alkynyl, further preferablyhydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl, and especiallypreferably methyl. The substituents for the substituted groups are eachindependently preferably a monovalent group selected from the groupconsisting of halogen (fluorine, chlorine, bromine or iodine), cyano,nitro, substituted or unsubstituted C₁-C₅ alkyl, substituted orunsubstituted C₂-C₅ alkenyl, substituted or unsubstituted C₂-C₅ alkynyl,substituted or unsubstituted C₃-C₆ cycloalkyl, substituted orunsubstituted C₃-C₆ cycloalkenyl, substituted or unsubstituted C₃-C₆cycloalkynyl, substituted or unsubstituted amino, and substituted orunsubstituted C₁-C₅ alkoxy, and more preferably a monovalent groupselected from the group consisting of halogen (fluorine, chlorine,bromine or iodine), cyano, nitro, unsubstituted C₁-C₅ alkyl,unsubstituted C₂-C₅ alkenyl, unsubstituted C₂-C₅ alkynyl, unsubstitutedC₃-C₆ cycloalkyl, unsubstituted C₃-C₆ cycloalkenyl, unsubstituted C₃-C₆cycloalkynyl, unsubstituted amino, and unsubstituted C₁-C₅ alkoxy. WhenR¹ is the groups, a compound represented by formula (I) can haveadrenomedullin activity substantially approximately equivalent to thatof natural adrenomedullin. Furthermore, the compound represented byformula (I) can sustainably exert adrenomedullin activity in a livingbody while substantially suppressing unwanted side effects.

L¹ is a m+1-valent linear or branched linking group. L¹ is preferably asubstituted or unsubstituted m+1-valent linear or branched hydrocarbongroup. The group optionally comprises one or more heteroatoms, analicyclic group, an aromatic group, an amide group (—CO—NH—), an estergroup (—CO—O—), or a urethane group (—O—CO—NH—). The substituents forthe substituted groups are each independently preferably a monovalentgroup selected from the group consisting of halogen (fluorine, chlorine,bromine or iodine), cyano, nitro, and a substituted or unsubstitutedlinear or branched hydrocarbon group.

L² and L^(2′) are each independently a bond or a divalent linking group.When each of L² and L^(2′) is a divalent linking group, L² and L^(2′)are each independently preferably a substituted or unsubstituteddivalent hydrocarbon group, an amide group (—CO—NH—), an ester group(—CO—O—), or a urethane group (—O—CO—NH—), and more preferablysubstituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted C₂-C₂₀ alkenylene, substituted or unsubstituted C₂-C₂₀alkynylene, substituted or unsubstituted C₃-C₂₀ cycloalkylene,substituted or unsubstituted C₄-C₂₀ cycloalkenylene, substituted orunsubstituted C₄-C₂₀ cycloalkynylene, substituted or unsubstituted 3- to6-membered heterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene, an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—). The groups optionally comprise one or more heteroatoms, anamide group (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—). The substituents for the substituted groups are eachindependently preferably a monovalent group selected from the groupconsisting of halogen (fluorine, chlorine, bromine or iodine), cyano,nitro, and a substituted or unsubstituted linear or branched hydrocarbongroup, and more preferably a monovalent group selected from the groupconsisting of halogen (fluorine, chlorine, bromine or iodine), cyano,nitro, unsubstituted C₁-C₅ alkyl, unsubstituted C₂-C₅ alkenyl,unsubstituted C₂-C₅ alkynyl, unsubstituted C₃-C₆ cycloalkyl,unsubstituted C₃-C₆ cycloalkenyl, unsubstituted C₃-C₆ cycloalkynyl,unsubstituted amino, and unsubstituted C₁-C₅ alkoxy.

When L¹, L² and L^(2′) are the groups, a compound represented by formula(I) can have adrenomedullin activity substantially approximatelyequivalent to that of natural adrenomedullin. Furthermore, the compoundrepresented by formula (I) can sustainably exert adrenomedullin activityin a living body while substantially suppressing unwanted side effects.

Suitable modifying group A is a modifying group represented by thefollowing formula (V), (VI), (VII) or (VIII):

In the formulas (V), (VI), (VII) and (VIII),

a is an integer of 1 or larger,

M³, M^(3′), M^(3″), M^(3′″) and M^(3″″) are each independently a bond ora PEG group, wherein when a plurality of M³, M^(3′), M^(3″), M^(3′″) orM^(3″″) are present, the plurality of M³, M^(3′), M^(3″), M^(3′″) orM^(3″″) are the same as or different from each other, and at least oneof M³, M^(3′), M^(3″), M^(3′″) and M^(3″″) is a PEG group,

R¹, R^(1′), R^(1″) and R^(1′″) are each independently hydrogen or amonovalent group,

R² is a bond or a divalent group,

R³, R^(3′) and R^(3″) are each independently a bond or divalent group,wherein when a plurality of R³, R^(3′) or R^(3″) are present, theplurality of R³, R^(3′) or R^(3″) are the same as or different from eachother, and

* is a binding position to the other moieties.

a is the number of repeats of a unit containing the PEG groups M³,M^(3′), M^(3″), M^(3′″) and M^(3″″). For example, when a is 1, the unithas no repeat structure. When a in the formula (V) is 2 or larger, theunit has a linear repeat structure. When a in the formulas (VI), (VII)and (VIII) is 2 or larger, the unit has a dendritically branched repeatstructure. a is typically an integer of 1 or larger and 5 or smaller andpreferably ranges from an integer from 1 to 5 and more preferably from 1to 2. When the number a of repeats of the unit containing the PEG groupsM³, M^(3′), M^(3″), M^(3′″) and M^(3″″) falls within the rangesdescribed above, the modifying group A comprising PEG groups can have alinear or branched structure.

When each of M³, M^(3′), M^(3″), M^(3′″) and M^(3″″) is a PEG group, thePEG group is typically a group represented by formula (III). The PEGgroup represented by formula (III) is as defined above. In this case, acompound represented by formula (I) can have adrenomedullin activitysubstantially approximately equivalent to that of naturaladrenomedullin. Furthermore, the compound represented by formula (I) cansustainably exert adrenomedullin activity in a living body whilesubstantially suppressing unwanted side effects.

R¹ is as defined above. R^(1′), R^(1″) and R^(1′″) are as defined in theR¹. In this case, a compound represented by formula (I) can haveadrenomedullin activity substantially approximately equivalent to thatof natural adrenomedullin. Furthermore, the compound represented byformula (I) can sustainably exert adrenomedullin activity in a livingbody while substantially suppressing unwanted side effects.

R² is preferably a bond, a substituted or unsubstituted divalenthydrocarbon group, an amide group (—CO—NH—), an ester group (—CO—O—), ora urethane group (—O—CO—NH—), and more preferably a bond, substituted orunsubstituted C₁-C₂₀ alkylene, substituted or unsubstituted C₂-C₂₀alkenylene, substituted or unsubstituted C₂-C₂₀ alkynylene, substitutedor unsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstitutedC₄-C₂₀ cycloalkenylene, substituted or unsubstituted C₄-C₂₀cycloalkynylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene, an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—). The divalent hydrocarbon group optionally comprises one ormore heteroatoms, an amide group (—CO—NH—), an ester group (—CO—O—), ora urethane group (—O—CO—NH—). The substituents for the substitutedgroups are each independently preferably a monovalent group selectedfrom the group consisting of halogen (fluorine, chlorine, bromine oriodine), cyano, nitro, substituted or unsubstituted C₁-C₅ alkyl,substituted or unsubstituted C₂-C₅ alkenyl, substituted or unsubstitutedC₂-C₅ alkynyl, substituted or unsubstituted C₃-C₆ cycloalkyl,substituted or unsubstituted C₃-C₆ cycloalkenyl, substituted orunsubstituted C₃-C₆ cycloalkynyl, substituted or unsubstituted amino,and substituted or unsubstituted C₁-C₅ alkoxy, and more preferably amonovalent group selected from the group consisting of halogen(fluorine, chlorine, bromine or iodine), cyano, nitro, unsubstitutedC₁-C₅ alkyl, unsubstituted C₂-C₅ alkenyl, unsubstituted C₂-C₅ alkynyl,unsubstituted C₃-C₆ cycloalkyl, unsubstituted C₃-C₆ cycloalkenyl,unsubstituted C₃-C₆ cycloalkynyl, unsubstituted amino, and unsubstitutedC₁-C₅ alkoxy. R² is preferably a bond or a substituted or unsubstitutedC₁-C₁₀ alkylene group, more preferably a bond, methylene, ethylene,propylene or butylene, and further preferably a bond or ethylene.

R³, R^(3′) and R^(3″) are each independently preferably a bond, asubstituted or unsubstituted divalent hydrocarbon group, an amide group(—CO—NH—), an ester group (—CO—O—), or a urethane group (—O—CO—NH—), andmore preferably a bond, substituted or unsubstituted C₁-C₂₀ alkylene,substituted or unsubstituted C₂-C₂₀ alkenylene, substituted orunsubstituted C₂-C₂₀ alkynylene, substituted or unsubstituted C₃-C₂₀cycloalkylene, substituted or unsubstituted C₄-C₂₀ cycloalkenylene,substituted or unsubstituted C₄-C₂₀ cycloalkynylene, substituted orunsubstituted 3- to 6-membered heterocycloalkylene, substituted orunsubstituted C₇-C₂₀ cycloalkylalkylene, substituted or unsubstituted 3-to 6-membered heterocycloalkyl-C₁-C₂₀ alkylene, substituted orunsubstituted C₄-C₂₀ arylene, substituted or unsubstituted C₅-C₂₀arylalkylene, substituted or unsubstituted 5- to 15-memberedheteroarylene, substituted or unsubstituted 5- to 15-memberedheteroaryl-C₁-C₂₀ alkylene, an amide group (—CO—NH—), an ester group(—CO—O—), or a urethane group (—O—CO—NH—). The divalent hydrocarbongroup optionally comprises one or more heteroatoms, an amide group(—CO—NH—), an ester group (—CO—O—), or a urethane group (—O—CO—NH—). Thesubstituents for the substituted groups are each independentlypreferably a monovalent group selected from the group consisting ofhalogen (fluorine, chlorine, bromine or iodine), cyano, nitro,substituted or unsubstituted C₁-C₅ alkyl, substituted or unsubstitutedC₂-C₅ alkenyl, substituted or unsubstituted C₂-C₅ alkynyl, substitutedor unsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted C₃-C₆cycloalkenyl, substituted or unsubstituted C₃-C₆ cycloalkynyl,substituted or unsubstituted amino, and substituted or unsubstitutedC₁-C₅ alkoxy, and more preferably a monovalent group selected from thegroup consisting of halogen (fluorine, chlorine, bromine or iodine),cyano, nitro, unsubstituted C₁-C₅ alkyl, unsubstituted C₂-C₅ alkenyl,unsubstituted C₂-C₅ alkynyl, unsubstituted C₃-C₆ cycloalkyl,unsubstituted C₃-C₆ cycloalkenyl, unsubstituted C₃-C₆ cycloalkynyl,unsubstituted amino, and unsubstituted C₁-C₅ alkoxy. R³, R^(3′) and R³are each independently preferably a bond, substituted or unsubstitutedC₁-C₁₀ alkylene group, a substituted or unsubstituted C₁-C₁₀ alkylenegroup containing an amide group, or an amide group (—CO—NH—), morepreferably a bond, methylene, ethylene, —CO—NH—(CH₂)₄—,—CH₂—O—CO—NH—(CH₂)₃— or —CO—NH—.

When each of R², R³, R^(3′) and R^(3″) is the groups, a compoundrepresented by formula (I) can have adrenomedullin activitysubstantially approximately equivalent to that of naturaladrenomedullin. Furthermore, the compound represented by formula (I) cansustainably exert adrenomedullin activity in a living body whilesubstantially suppressing unwanted side effects.

Especially suitable modifying group A is a modifying group representedby the following formula (V-1-1), (VI-1-1), (VII-1-1), (VII-1-2),(VII-2-1), or (VIII-1-1):

wherein

n is as defined above,

n′ is as defined above about n, and

* is a binding position to the other moieties.

In the formula (V-1-1), the PEG group preferably has a weight-averagemolecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa or 80kDa in total.

In the formula (VI-1-1), the PEG groups preferably have a weight-averagemolecular weight of 40 kDa in total.

In the formula (VII-1-1), the PEG groups preferably have aweight-average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40kDa, 60 kDa or 80 kDa in total.

In the formula (VII-1-2), the PEG groups preferably have aweight-average molecular weight of 50 kDa in total. In this case,typically, the ethylene oxide unit of (CH₂CH₂O)_(n) has a weight-averagemolecular weight of 40 kDa in total, and the ethylene oxide unit of(CH₂CH₂O)_(n′) has a weight-average molecular weight of 10 kDa in total.

In the formula (VII-2-1), the PEG groups preferably have aweight-average molecular weight of 40 kDa in total. In this case,typically, the ethylene oxide unit of (CH₂CH₂O) has a weight-averagemolecular weight of 30 kDa in total, and the ethylene oxide unit of(CH₂CH₂O)_(n′) has a weight-average molecular weight of 10 kDa in total.Alternatively, the PEG groups preferably have a weight-average molecularweight of 60 kDa in total. In this case, typically, the ethylene oxideunit of (CH₂CH₂O)_(n) has a weight-average molecular weight of 50 kDa intotal, and the ethylene oxide unit of (CH₂CH₂O)_(n′) has aweight-average molecular weight of 10 kDa in total. Alternatively, thePEG groups preferably have a weight-average molecular weight of 80 kDain total. In this case, typically, the ethylene oxide unit of(CH₂CH₂O)_(n) has a weight-average molecular weight of 70 kDa in total,and the ethylene oxide unit of (CH₂CH₂O)_(n′) has a weight-averagemolecular weight of 10 kDa in total.

In the formula (VIII-1-1), the PEG groups preferably have aweight-average molecular weight of 40 kDa in total.

By use of the groups as the modifying group A, a compound represented byformula (I) can substantially suppress unwanted side effects andsustainably exert adrenomedullin activity in a living body, whilemaintaining pharmacological effects of natural adrenomedullin.

In the formula (I), the peptide moiety B is required to be linked to theother moieties through a covalent bond of the nitrogen atom of theN-terminal α-amino group of the peptide moiety B to the carbon atom ofthe methylene group. In the invention, a compound in which the modifyinggroup A comprising one or more PEG groups and the peptide moiety B arelinked in the manner of linking described above may be described as“alkylamine linkage-type adrenomedullin derivative”. The alkylaminelinkage-type adrenomedullin derivative has higher adrenomedullinactivity as compared to an adrenomedullin derivative in whichadrenomedullin is linked to the other moieties through an amide bond ofthe nitrogen atom of the N-terminal α-amino group of the adrenomedullin(hereinafter, also described as “amide linkage-type adrenomedullinderivative”), as with the adrenomedullin derivative described in NonPatent Literature 10. Furthermore, the alkylamine linkage-typeadrenomedullin derivative of the invention represented by the formula(I) further suppresses unwanted side effects (such as excessivedecreased blood pressure, tachycardia associated with increased reflexsympathetic nerve activity, and/or increased activity of renin) ascompared to the amide linkage-type adrenomedullin derivative. Therefore,the compound of the invention represented by formula (I) can sustainablyexert adrenomedullin activity in a living body while further suppressingunwanted side effects, as compared to known adrenomedullin derivatives.

In an especially suitable compound represented by formula (I), A is amodifying group comprising PEG groups, represented by formula (V-1-1),(VI-1-1), (VII-1-1), (VII-1-2), (VII-2-1), or (VIII-1-1), and

B is a peptide moiety derived from adrenomedullin or a modified formthereof with adrenomedullin activity, the adrenomedullin or the modifiedform being a peptide selected from the group consisting of:

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, ora peptide consisting of the amino acid sequence of SEQ ID NO: 1 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, ora peptide consisting of the amino acid sequence of SEQ ID NO: 3 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, ora peptide consisting of the amino acid sequence of SEQ ID NO: 5 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, ora peptide consisting of the amino acid sequence of SEQ ID NO: 9 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, ora peptide consisting of the amino acid sequence of SEQ ID NO: 11 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(i) any peptide of (a) to (f) wherein the peptide is amidated at theC-terminus thereof; and

(j) any peptide of (a) to (f) wherein the peptide has a glycine residueadded to the C-terminus thereof, or a peptide selected from the groupconsisting of:

(h′) any peptide of (a) to (d) wherein the peptide has deletion of aminoacid residues at positions 1 to 15, positions 1 to 10, or positions 1 to5 from the N-terminus thereof and has adrenomedullin activity, or thepeptide of (e) or (f) wherein the peptide has deletion of amino acidresidues at positions 1 to 13, positions 1 to 8, or positions 1 to 5from the N-terminus thereof and has adrenomedullin activity;(i) the peptide of (h′) wherein the peptide is amidated at theC-terminus thereof; and(j) the peptide of (h′) wherein the peptide has a glycine residue addedto the C-terminus thereof. A compound represented by formula (I) havingthe properties described above can substantially suppress unwanted sideeffects and sustainably exert adrenomedullin activity in a living body,while maintaining pharmacological effects of natural adrenomedullin.

Another aspect of the invention relates to a compound represented byformula (X):A′-CO—B  (X)or a salt thereof, or a hydrate thereof. In the present specification,the compound represented by formula (X) may be described as “urethanelinkage-type adrenomedullin derivative”.

In the formula (X), B is required to be a peptide moiety derived fromadrenomedullin or a modified form thereof with adrenomedullin activity.The peptide moiety B is as defined above about the compound representedby formula (I).

A′ is required to be a modifying group comprising one or more PEGgroups. However, A′ is required to be linked to the other moietiesthrough a covalent bond of the oxygen atom of the modifying groupcomprising PEG groups to the carbon atom of the carbonyl group. Themodifying group A′ having such a structure allows a compound representedby formula (X) to have a structure having the modifying group A′ and thepeptide moiety B linked via a urethane bond.

A′ is preferably a modifying group represented by the following formula(XI), (XI′) or (XII):

In the formulas (XI), (XI′) and (XII), * is a binding position to theother moieties.

In the formulas (XI), (XI′) and (XII), a, R¹, R^(1′), R², R³, R^(3′),R^(3″), M¹, M³, M^(3′) and M^(3″) are as defined above about thecompound represented by formula (I).

Especially suitable modifying group A′ is a modifying group representedby the following formula (XI-1-1), (XII-1-1) or (XII-2-1):

wherein

n is as defined above,

n′ is as defined above about n, and

* is a binding position to the other moieties.

In the formula (XI-1-1), the PEG group preferably has a weight-averagemolecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa or 80kDa in total.

In the formula (XII-1-1), the PEG groups preferably have aweight-average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40kDa, 60 kDa or 80 kDa in total.

In the formula (XII-2-1), the PEG groups preferably have aweight-average molecular weight of 40 kDa in total. In this case,typically, the ethylene oxide unit of (CH₂CH₂O)_(n) has a weight-averagemolecular weight of 30 kDa in total, and the ethylene oxide unit of(CH₂CH₂O)_(n′) has a weight-average molecular weight of 10 kDa in total.Alternatively, the PEG groups preferably have a weight-average molecularweight of 60 kDa in total. In this case, typically, the ethylene oxideunit of (CH₂CH₂O)_(n) has a weight-average molecular weight of 50 kDa intotal, and the ethylene oxide unit of (CH₂CH₂O)_(n′) has aweight-average molecular weight of 10 kDa in total. Alternatively, thePEG groups preferably have a weight-average molecular weight of 80 kDain total. In this case, typically, the ethylene oxide unit of(CH₂CH₂O)_(n) has a weight-average molecular weight of 70 kDa in total,and the ethylene oxide unit of (CH₂CH₂O)_(n′) has a weight-averagemolecular weight of 10 kDa in total.

By use of the groups as the modifying group A′, a compound representedby formula (X) can sustainably exert adrenomedullin activity in a livingbody while maintaining pharmacological effects of naturaladrenomedullin.

In formula (X), the peptide moiety B is required to be linked to theother moieties through a covalent bond of the nitrogen atom of theN-terminal α-amino group of the peptide moiety B to the carbon atom ofthe carbonyl group. The urethane linkage-type adrenomedullin derivativehas higher adrenomedullin activity as compared to the amide linkage-typeadrenomedullin derivative described in Non Patent Literature 10.Therefore, the compound of the invention represented by formula (X) cansustainably exert higher adrenomedullin activity in a living body ascompared to known adrenomedullin derivatives.

In an especially suitable compound represented by formula (X),

A′ is a modifying group comprising PEG groups, represented by formula(XI-1-1), (XII-1-1) or (XII-2-1), and

B is a peptide moiety derived from adrenomedullin or a modified formthereof with adrenomedullin activity, the adrenomedullin or the modifiedform being a peptide selected from the group consisting of:

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, ora peptide consisting of the amino acid sequence of SEQ ID NO: 1 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, ora peptide consisting of the amino acid sequence of SEQ ID NO: 3 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, ora peptide consisting of the amino acid sequence of SEQ ID NO: 5 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21;

(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, ora peptide consisting of the amino acid sequence of SEQ ID NO: 9 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, ora peptide consisting of the amino acid sequence of SEQ ID NO: 11 andhaving a disulfide bond formed by the cysteine residues at positions 14and 19;

(i) any peptide of (a) to (f) wherein the peptide is amidated at theC-terminus thereof; and

(j) any peptide of (a) to (f) wherein the peptide has a glycine residueadded to the C-terminus thereof, or a peptide selected from the groupconsisting of:

(h′) any peptide of (a) to (d) wherein the peptide has deletion of aminoacid residues at positions 1 to 15, positions 1 to 10, or positions 1 to5 from the N-terminus thereof and has adrenomedullin activity, or thepeptide of (e) or (f) wherein the peptide has deletion of amino acidresidues at positions 1 to 13, positions 1 to 8, or positions 1 to 5from the N-terminus thereof and has adrenomedullin activity;(i) the peptide of (h′) wherein the peptide is amidated at theC-terminus thereof; and(j) the peptide of (h′) wherein the peptide has a glycine residue addedto the C-terminus thereof. A compound represented by formula (X) havingthe properties described above can sustainably exert higheradrenomedullin activity in a living body as compared to knownadrenomedullin derivatives.

In the invention, compounds represented by formulas (I) and (X) includenot only the compounds themselves but also salts thereof. When compoundsrepresented by formulas (I) and (X) are in the form of salt, they arepreferably pharmaceutically acceptable salts. Counterions in salts ofthe compounds of the invention preferably include, but are not limitedto, for example, cations such as a sodium, potassium, calcium,magnesium, or substituted or unsubstituted ammonium ion, or anions suchas a chloride, bromide, iodide, phosphate, nitrate, sulfate, carbonate,bicarbonate, perchlorate, formate, acetate, trifluoroacetate,propionate, lactate, maleate, hydroxymaleate, methylmaleate, fumarate,adipate, benzoate, 2-acetoxybenzoate, p-aminobenzoate, nicotinate,cinnamate, ascorbate, pamoate, succinate, salicylate,bismethylenesalicylate, oxalate, tartrate, malate, citrate, gluconate,aspartate, stearate, palmitate, itaconate, glycolate, glutamate,benzenesulfonate, cyclohexylsulfamate, methanesulfonate,ethanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate, ornaphthalenesulfonate ion. When compounds represented by formulas (I) and(X) are in the form of salt with any of the counterions, adrenomedullinactivity of the compounds can be substantially approximately equivalentto that of natural adrenomedullin.

Compounds represented by formulas (I) and (X) include not only thecompounds themselves but also solvates of the compounds or saltsthereof. When compounds represented by formulas (I) and (X) or saltsthereof are in the form of solvate, they are preferably pharmaceuticallyacceptable solvates. Solvents that can form solvates with the compoundsor salts thereof include, but are not limited to, for example, water ororganic solvents such as methanol, ethanol, 2-propanol (isopropylalcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine,acetonitrile, or ethyl acetate. When compounds represented by formulas(I) and (X) or salts thereof are in the form of solvate with any of thesolvents described above, adrenomedullin activity of the compounds canbe substantially approximately equivalent to that of naturaladrenomedullin.

Compounds represented by formulas (I) and (X) include not only thecompounds themselves described above or below but also protected formsthereof. In the present specification, a “protected form” means a formin which any suitable protecting group is introduced into one or morefunctional groups (such as a side-chain amino group of lysine residue)of the compound. In the present specification, a “protecting group”means a group that is introduced into a specific functional group toprevent any unwanted reaction from proceeding, will be removedquantitatively under a specific reaction condition, and is substantiallystable, or inactive, under any reaction condition other than thespecific reaction condition. Protecting groups that can form protectedforms of the compounds include, but are not limited to, for example,t-butoxycarbonyl (Boc), 2-bromobenzyloxycarbonyl (BrZ),9-fluorenylmethoxycarbonyl (Fmoc), p-toluenesulfonyl (Tos), benzyl(Bzl), 4-methylbenzyl (4-MeBzl), 2-chlorobenzyloxycarbonyl (ClZ),cyclohexyl (cHex), and phenacyl (Pac); other protecting groups of aminogroups include benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, benzhydryloxycarbonyl,2-(p-biphenyl)isopropyloxycarbonyl,2-(3,5-dimethoxyphenyl)isopropyloxycarbonyl,p-phenylazobenzyloxycarbonyl, triphenylphosphonoethyloxycarbonyl,9-fluorenylmethyloxycarbonyl, t-amyloxyoxycarbonyl,diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethyloxycarbonyl,allyloxycarbonyl, 2-methylsulfonylethyl oxycarbonyl,2,2,2-trichloroethyloxycarbonyl, cyclopentyloxycarbonyl,cyclohexyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl,benzenesulfonyl, mesitylenesulfonyl, methoxytrimethylphenylsulfonyl,2-nitrobenzensulfonyl, 2-nitrobenzenesulfenyl, 4-nitrobenzensulfonyl,and 4-nitrobenzene sulfenyl; other protecting groups of carboxyl groupsinclude methyl esters, ethyl esters, t-butyl esters, p-methoxybenzylesters, and p-nitrobenzyl esters; other side-chain protecting groups ofArg include 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl,4-methoxy-2,3,6-trimethylbenzenesulphonyl,2,2,5,7,8-pentamethylchroman-6-sulfonyl, and 2-methoxybenzenesulfonyl;other protecting groups of Tyr include 2,6-dichlorobenzyl, t-butyl, andcyclohexyl; other protecting groups of Cys include 4-methoxybenzyl,t-butyl, trityl, acetamidomethyl, and 3-nitro-2-pyridine sulfenyl; otherprotecting groups of His include benzyloxymethyl,p-methoxybenzyloxymethyl, t-butoxymethyl, trityl, and 2,4-dinitrophenyl;and other protecting groups of Ser and Thr include t-butyl. When acompound represented by formulas (I) and (X) is in a protected form withany of the protecting groups described above, adrenomedullin activity ofthe compound can be substantially approximately equivalent to that ofnatural adrenomedullin.

Compounds represented by formulas (I) and (X) include individualenantiomer and diastereomer of the compounds, and mixtures ofstereoisomeric forms of the compounds such as racemates.

A compound represented by formula (I) and (X) having the propertiesdescribed above can substantially suppress unwanted side effects andsustainably exert adrenomedullin activity in a living body, whilemaintaining pharmacological effects of natural adrenomedullin.

<2. Pharmaceutical Use of Adrenomedullin Derivatives>

A compound of the invention represented by formulas (I) and (X) cansustainably exert bioactivity substantially approximately equivalent tothat of adrenomedullin, which is the parent molecule of the compound,(i.e., adrenomedullin activity) in a living body. Therefore, theinvention relates to a medicament comprising a compound of the inventionrepresented by formula (I) or (X) or a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable hydrate thereof as an activeingredient.

A compound of the invention represented by formulas (I) and (X) may beused alone or in combination with one or more pharmaceuticallyacceptable components when the compound is applied to pharmaceuticaluse. A medicament of the invention can be formulated into various dosageforms commonly used in the art depending on the desired mode ofadministration. Thus, the medicament of the invention can also beprovided in the form of a pharmaceutical composition comprising acompound of the invention represented by formulas (I) and (X) and one ormore pharmaceutically acceptable carriers. Pharmaceutical compositionsof the invention may comprise, in addition to the components describedabove, one or more pharmaceutically acceptable carriers, excipients,binders, vehicles, dissolution aids, preservatives, stabilizers, bulkingagents, lubricants, surfactants, oily liquids, buffering agents,soothing agents, antioxidants, sweetening agents, flavoring agents, andso forth.

Dosage forms of medicaments comprising a compound of the inventionrepresented by formulas (I) and (X) as an active ingredient are notparticularly limited and may be a formulation for parenteral or oraladministration. Dosage forms of medicaments of the invention may also bea formulation in unit dosage form or in multiple dosage form.Formulations for use in parenteral administration include, for example,injections such as sterile solutions or suspensions in water or anyother pharmaceutically acceptable liquid. Additive agents that can beadmixed into the injections include, but are not limited to, forexample, vehicles such as physiological saline and isotonic solutionscomprising glucose or other pharmaceutic aids (such as D-sorbitol,D-mannitol, or sodium chloride); dissolution aids such as alcohols (suchas ethanol or benzyl alcohol), esters (such as benzyl benzoate), andpolyalcohols (such as propylene glycol or polyethylene glycol); nonionicsurfactants such as polysorbate 80 or polyoxyethylene hydrogenatedcastor oil; oily liquids such as sesame oil or soybean oil; bufferingagents such as phosphate buffer or sodium acetate buffer; soothingagents such as benzalkonium chloride or procaine hydrochloride;stabilizers such as human serum albumin or polyethylene glycol;preservatives; and antioxidants. The prepared injection will begenerally filled in any suitable vial (such as an ampule) and preservedunder an appropriate environment until use.

The formulations for use in oral administration include, for example, atablet optionally coated with sugar coating or soluble film, a capsule,an elixir, a microcapsule, a tablet, a syrup, and a suspension. Additiveagents that can be admixed into tablets or capsules and so forthinclude, but are not limited to, for example, binders such as gelatin,cornstarch, gum tragacanth, and gum arabic; excipients such ascrystalline cellulose; bulking agents such as cornstarch, gelatin, andalginate; lubricants such as magnesium stearate; sweetening agents suchas sucrose, lactose, or saccharin; and flavoring agents such aspeppermint, Gaultheria adenothrix oil, or cherry. A formulation mayfurther include liquid carriers such as oils/fats when the formulationis in the form of a capsule.

The compound of the invention represented by formulas (I) and (X) cansustainably exert adrenomedullin activity substantially approximatelyequivalent to that of adrenomedullin, which is the parent molecule ofthe compound, in a living body. Thus, a medicament comprising a compoundof the invention represented by formulas (I) and (X) as an activeingredient can be formulated into a depot formulation. In this case, themedicament of the invention in the dosage form of depot formulation can,for example, be implanted subcutaneously or intramuscularly oradministered by intramuscular injection. The depot formulation of themedicament of the invention allows the compound of the inventionrepresented by formulas (I) and (X) to sustainably exert adrenomedullinactivity for a long period of time.

The medicament comprising a compound of the invention represented byformulas (I) and (X) as an active ingredient can be combined with one ormore other drugs useful as medicaments. In this case, the medicament ofthe invention may be provided in the form of a single medicamentcomprising the compound of the invention represented by formula (I) or(X) or a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable hydrate thereof and one or more other drugs, or may beprovided in the form of a medicament combination or kit comprising aplurality of formulations into which the compound of the inventionrepresented by formula (I) or (X) or a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable hydrate thereof and one ormore other drugs are separately formulated. For the medicamentcombination or kit, each formulation can be administered simultaneouslyor separately (such as sequentially).

For applying compounds of the invention represented by formulas (I) and(X) to pharmaceutical use, the compounds represented by formulas (I) and(X) include not only the compounds themselves but also pharmaceuticallyacceptable salts thereof and pharmaceutically acceptable solvatesthereof. The pharmaceutically acceptable salts of compounds of theinvention represented by formulas (I) and (X) and pharmaceuticallyacceptable solvates thereof preferably include, but are not limited to,for example, salts or solvates exemplified above. When compoundsrepresented by formulas (I) and (X) are in the form of any of the saltsor solvates described above, the compounds can be applied to the desiredpharmaceutical use.

A medicament comprising a compound of the invention represented byformulas (I) and (X) as an active ingredient can prevent or treatvarious conditions, diseases, and/or disorders that will be prevented ortreated with adrenomedullin. The conditions, diseases, and/or disordersinclude, but are not limited to, for example, the following:

(1) Cardiovascular diseases: cardiac insufficiency, pulmonaryhypertension, arteriosclerosis obliterans, Buerger's disease, myocardialinfarction, lymphedema, Kawasaki's disease, myocarditis, high bloodpressure, organ dysfunctions due to high blood pressure, andarteriosclerosis.

(2) Kidney and water and electrolyte system disorders: kidney failureand nephritis.

(3) Brain and nervous system diseases: cerebral infarction, dementia,and encephalitis.

(4) Urogenital diseases: erectile dysfunction (ED).

(5) Gastrointestinal diseases: inflammatory bowel disease, ulcerativedisease, intestinal Behcet's disease, and hepatic failure.

(6) Orthopedic disease: arthritis.

(7) Endocrine metabolic disease: diabetes and organ dysfunctions due todiabetes, and primary aldosteronism.

(8) Others: septic shock, auto-immune disease, multiple organ failure,pressure sore, wound healing, and alopecia.

The cardiovascular disease is preferably any of myocardial infarction,pulmonary hypertension, and cardiac insufficiency. The gastrointestinaldisease is preferably any of inflammatory diseases including asteroid-resistant or steroid-dependent inflammatory bowel disease (suchas ulcerative colitis, Crohn's disease, or intestinal tract Behcet'sdisease).

A compound of the invention represented by formulas (I) and (X) has astructure in which adrenomedullin, which is a natural bioactive peptide,is linked to a modifying group. This structure allows the compound ofthe invention represented by formulas (I) and (X) to be safe and havelow toxicity. Therefore, the medicament comprising the compound of theinvention represented by formulas (I) and (X) as an active ingredientcan be applied to various subjects in need of prevention or treatment ofthe condition, disease, and/or disorder. The subjects are preferablyhuman or non-human mammalian (such as warm-blooded animal including pig,dog, cattle, rat, mouse, guinea pig, rabbit, chicken, sheep, cat,monkey, hamadryas baboon, or chimpanzee) subjects or patients. Themedicament of the invention can be administered to the subjects toprevent or treat various conditions, diseases, and/or disorders thatwill be prevented or treated with adrenomedullin.

In the present specification, “prevention” means that onset (developmentor occurrence) of a condition, disease, and/or disorder will besubstantially precluded. On the other hand, in the presentspecification, “treatment” means suppression (such as suppression ofprogression), remission, restoration, and/or cure of a condition,disease, and/or disorder that has appeared (developed or occurred).

The compound of the invention represented by formulas (I) and (X) can beused to prevent or treat the condition, disease, and/or disorderdescribed above (such as a cardiovascular disease, peripheral vasculardisease, or inflammatory disease) in subjects with the condition,disease, and/or disorder. Therefore, the medicament of the invention ispreferably a medicament for use in the prevention or treatment of thecondition, disease, and/or disorder described above and is morepreferably a medicament for use in the prevention or treatment of acardiovascular disease, an inflammatory disease, or a peripheralvascular disease. The invention also relates to an agent for preventingor treating a cardiovascular disease, an inflammatory disease, or aperipheral vascular disease comprising a compound of the inventionrepresented by formula (I) or (X) or a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable hydrate thereof as an activeingredient. The compound of the invention represented by formulas (I)and (X) can be used to prevent or treat the condition, disease, and/ordisorder described above to sustainably prevent or treat the condition,disease, and/or disorder.

Compounds of the invention represented by formulas (I) and (X) can beused to prevent or treat the condition, disease, and/or disorderdescribed above (such as a cardiovascular disease, peripheral vasculardisease, or inflammatory disease) in subjects with the condition,disease, and/or disorder. Therefore, one embodiment of the invention isa method for preventing or treating the disease or condition describedabove, comprising administering an effective amount of a compound of theinvention represented by formulas (I) and (X) or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable hydratethereof to a subject in need of prevention or treatment of thecondition, disease, and/or disorder described above. The condition,disease, and/or disorder is preferably any of cardiovascular diseases,peripheral vascular diseases, and inflammatory diseases. Compounds ofthe invention represented by formulas (I) and (X) can be administered tosubjects in need of prevention or treatment of the condition, disease,and/or disorder to prevent or treat the condition, disease, and/ordisorder.

Another embodiment of the invention is a compound of the inventionrepresented by formula (I) or (X) or a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable hydrate thereof for use in theprevention or treatment of the condition, disease, and/or disorderdescribed above. An alternative embodiment of the invention is use of acompound of the invention represented by formula (I) or (X) or apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable hydrate thereof for manufacturing a medicament for theprevention or treatment of the condition, disease, and/or disorderdescribed above. The condition, disease, and/or disorder is preferablyany of cardiovascular diseases, inflammatory diseases, and peripheralvascular diseases. The medicament of the invention can be used toprevent or treat the condition, disease, and/or disorder described aboveto sustainably prevent or treat the condition, disease, and/or disorder.

When a medicament comprising a compound of the invention represented byformulas (I) and (X) as an active ingredient is administered to asubject, particularly a human patient, the precise dose and number ofdoses will be determined considering many factors including age and sexof the subject, the precise condition (such as severity) of thecondition, disease, and/or disorder to be prevented or treated, and theroute of administration. The therapeutically effective dose and numberof doses should be ultimately determined by the attending physician.Therefore, the compound represented by formulas (I) and (X), which is anactive ingredient in the medicament of the invention, will beadministered to the subject in the therapeutically effective dose andnumber of doses. For example, when the medicament of the invention isadministered to a human patient, a dose of the compound represented byformulas (I) and (X), which is an active ingredient, will usually rangefrom 0.01 to 100 mg per 60 kg of body weight per day and typically from0.01 to 10 mg per 60 kg of body weight per day.

Route of administration and number of doses of a medicament comprising acompound of the invention represented by formulas (I) and (X) as anactive ingredient are not particularly limited and the medicament may beadministered orally or parenterally in a single dose or in multipledoses. The medicament of the invention is preferably administeredparenterally such as intravenously, by intestinal infusion,subcutaneously, intramuscularly, or intraperitoneally, and morepreferably intravenously or subcutaneously. The medicament of theinvention is also preferably administered in a single dose. Themedicament of the invention is particularly preferably used inintravenous or subcutaneous administration in a single dose.Adrenomedullin, which is the parent molecule of compounds of theinvention represented by formulas (I) and (X), has a strong vasodilatoryeffect. This strong vasodilatory effect may cause unwanted side effectssuch as excessive decreased blood pressure, tachycardia associated withincreased reflex sympathetic nerve activity, and/or increased activityof renin when a therapeutically effective amount of adrenomedullin isadministered in a single dose. On the other hand, compounds of theinvention represented by formulas (I) and (X) can significantly prolongblood half-life as compared to natural adrenomedullin while retainingadrenomedullin activity substantially approximately equivalent to thatof natural adrenomedullin. Therefore, intravenous administration of themedicament comprising a compound of the invention represented byformulas (I) and (X) as an active ingredient to a subject in a singledose allows the medicament to sustainably prevent or treat a condition,disease, and/or disorder in the subject while suppressing unwanted sideeffects due to the vasodilation effect of adrenomedullin.

<3. Method for Producing Adrenomedullin Derivatives>

The invention also relates to a method for producing a compound of theinvention represented by formulas (I) and (X).

[3-1. Step of Preparing Precursors]

The method of the invention may comprise preparing at least any of aprecursor of peptide moiety B derived from adrenomedullin or a modifiedform thereof and a precursor of modifying group A or A′ comprising oneor more polyethylene glycol groups.

In the invention, “a precursor of peptide moiety B derived fromadrenomedullin or a modified form thereof” means the adrenomedullin orthe modified form thereof or means a derivative of the peptide moiety Bthat has been suitably modified or activated so that the peptide moietyB and the modifying group A or A′ are linked together via condensationreactions in the linking step as described below. The precursor ofpeptide moiety B is preferably adrenomedullin or a modified form thereofitself, or a protected form thereof.

The precursor of modifying group A is typically a precursor aldehyde ofmodifying group A comprising one or more polyethylene glycol groups, theprecursor aldehyde being represented by formula (I-1):A-CHO  (I-1)Preparation of precursors having properties described above in this stepallows high-yield formation of a compound represented by formula (I)through reactions of linking each precursor in the linking stepdescribed below.

The precursor of modifying group A′ is typically a precursorp-nitrophenyl carbonate ester of modifying group A′ comprising one ormore polyethylene glycol groups, the precursor p-nitrophenyl carbonateester being represented by formula (X-1):A′-CO—O—C₆H₄-p—NO₂  (X-1)Alternatively, the precursor of modifying group A′ may be a precursorN-hydroxysuccinimidyl carbonate ester of modifying group A′ comprisingone or more polyethylene glycol groups, the precursorN-hydroxysuccinimidyl carbonate ester being represented by formula(X-2):A′-CO—O—C₄H₄NO₂  (X-2)Preparation of precursors having properties described above in this stepallows high-yield formation of a compound represented by formula (X)through reactions of linking each precursor in the linking stepdescribed below.

In this step, the precursor of peptide moiety B derived fromadrenomedullin or a modified form thereof can be prepared by any meanscommonly used in the art. The means may be, for example, a peptidesynthesis method on solid phase system or in liquid phase system, or amethod for purifying natural peptides from human or non-human mammaliantissues or cells that can produce adrenomedullin when the precursor ofpeptide moiety B is adrenomedullin or a modified form thereof itself.Alternatively, the means may be a method for overexpression of arecombinant protein using DNA encoding adrenomedullin in human ornon-human mammal that can produce adrenomedullin (such as SEQ ID NO: 2,4, 6, 8, 10, or 12) in a transformation system such as Escherichia colior Saccharomyces cerevisiae. Alternatively, the already producedpeptides may be also purchased. Any case will be included in theembodiment of this step.

A precursor that has a disulfide bond formed by two cysteine residues inthe amino acid sequence in the precursor of peptide moiety B prepared bythe means described above can be obtained by disulfide bond formationbetween thiol groups of two cysteine residues in the amino acidsequence. A precursor in which the disulfide bond formed between twocysteine residues in the amino acid sequence of the precursor of peptidemoiety B prepared by the means described above has been substituted withan ethylene group can be obtained by substitution of the disulfide bondwith an ethylene group. The formation reaction of a disulfide bond andthe substitution reaction with an ethylene group can be performed basedon any condition commonly used in the art. The formation reaction of adisulfide bond and the substitution reaction with an ethylene group maybe performed in this step or in the linking step described below. Anycase will be included in the embodiment of the step.

When at least any of the precursor of peptide moiety B, and theprecursor of modifying group A or A′ are in a protected form, theprotection step in which one or more protecting groups are introducedinto at least any of the precursor of peptide moiety B, and theprecursor of modifying group A or A′ and/or the deprotection step inwhich at least any of one or more protecting groups in protected formsof the precursor of peptide moiety B, and the precursor of modifyinggroup A or A′ are deprotected may be performed in this step as desired.The protection and deprotection steps can be performed with anyprotection and deprotection reaction commonly used in the art. Theprotection and deprotection steps may be performed in this step or inthe linking step described below. Any case will be included in theembodiment of this step.

[3-2. Linking Step]

The method of the invention is required to comprise a linking step oflinking the precursor of peptide moiety B derived from adrenomedullin ora modified form thereof, and the precursor of modifying group A or A′ togive a compound represented by formula (I) or (X).

To form a compound represented by formula (I), this step is typicallyperformed by reacting the precursor of peptide moiety B with theprecursor aldehyde represented by formula (I-1) of modifying group Acomprising one or more PEG groups in the presence of a reducing agent.Reducing agents that can be used in this step can include, but are notlimited to, sodium cyanoborohydride (NaCNBH₃), sodium borohydride(NaBH₄), dimethylamine borate, trimethylamine borate, pyridine borate,pyridine borane, 2-picoline borane and 3-picoline borane. The reactiontemperature in this step preferably ranges from −20 to 50° C. and morepreferably from 0 to 15° C. The reaction time in this step preferablyranges from 5 minutes to 100 hours.

To form a compound represented by formula (X), this step is typicallyperformed by reacting the precursor of peptide moiety B with theprecursor p-nitrophenyl carbonate ester or N-hydroxysuccinimidylcarbonate ester represented by formula (X-1) or (X-2) of modifying groupA′ comprising one or more PEG groups in the presence of a base. Basesthat can be used in this step can include, but are not limited to,triethylamine, pyridine and dimethylaminopyridine. The reactiontemperature in this step preferably ranges from 0 to 50° C. The reactiontime in this step preferably ranges from 5 minutes to 200 hours.

EXAMPLES

Hereinafter, the present invention will be described furtherspecifically with reference to Examples. However, the technical scope ofthe present invention is not intended to be limited by these Examples.

Experiment I: Preparation of Full-Length Adrenomedullin DerivativeExperiment I-1: Synthesis of Full-Length Adrenomedullin DerivativeExperiment I-1-1: Synthesis of CH₃O-PEG (5k)-(CH₂)₅—CO—^(α)NH-(h.AM(1-52)) (Compound (1))

In accordance with the method described in the known literature (Kubo, Ket al., “Biological properties of adrenomedullin conjugated withpolyethylene glycol.”, Peptides, 2014, vol. 57, p. 118-21), apolyethylene glycol group with a weight-average molecular weight of 5kDa (hereinafter, also described as “PEG (5k)”) was linked via an amidebond to the N-terminal amino group of a Cys¹⁶-Cys²¹ disulfide bridgeform of a peptide having the amino acid sequence ofH-Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “h.AM (1-52)”), which was a peptidecorresponding to amino acid residues 1 to 52 of human adrenomedullin(SEQ ID NO: 1), using a N-hydroxysuccinimide active ester-typeCH₃O-PEGylation reagent (PEG-1) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₅—CO—O—NHS) of5 kDa to synthesize an amide linkage-type PEG (5k) adrenomedullinderivative (CH₃O-PEG (5k)-(CH₂)₅—CO—^(α)NH-(h.AM (1-52))) (1).

Experiment I-1-2: Synthesis of CH₃O-PEG (20k)-(CH₂)₅—CO—^(α)NH-(h.AM(1-52)) (Compound (2))

In the same way as in experiment I-1-1, a polyethylene glycol group witha weight-average molecular weight of 20 kDa (hereinafter, also describedas “PEG (20k)”) was linked via an amide bond to the N-terminal aminogroup of the h.AM (1-52) peptide using a N-hydroxysuccinimide activeester-type CH₃O-PEGylation reagent (PEG-1)(CH₃O—(CH₂CH₂O)_(n)—(CH₂)₅—CO—O—NHS) of 20 kDa to synthesize an amidelinkage-type PEG (20k) adrenomedullin derivative (CH₃O-PEG(20k)-(CH₂)₅—CO—^(α)NH-(h.AM (1-52))) (2).

Experiment I-1-3: Synthesis of CH₃O-PEG (10k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52)) (Compound (3))

2 mg of the h.AM (1-52) peptide was dissolved in a 100 mM sodium acetatebuffer (pH 5.5) to obtain 2 mL of a peptide solution. To this peptidesolution, 16 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-2)(CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecular weightof 10 kDa was added under ice cooling. To this peptide solution, NaCNBH₃was further added so as to attain a final concentration of 20 mM. Thereaction solution was left at 4° C. for 24 hours. The obtained reactionsolution was diluted 5-fold with a 50 mM sodium acetate buffer (pH 4.0).The diluted reaction solution was applied at a flow rate of 2 mL/hr toSP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL) equilibratedwith a 50 mM sodium acetate buffer (pH 4.0). The column was washed with2 mL of a 50 mM sodium acetate buffer (pH 4.0). Subsequently, 5 mL of a50 mM sodium acetate buffer (pH 5.0) containing 1 M NaCl was applied tothe column to obtain an eluted fraction. An alkylamine linkage-type PEG(10k) adrenomedullin derivative (CH₃O-PEG (10k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52))) (3) and unreacted h.AM (1-52) peptide were recovered in theeluted fraction. This eluted fraction was concentrated into 0.2 mL usingan ultrafiltration membrane (Amicon Ultra 4, Merck Millipore). Theobtained concentrate was purified and fractionated using ahigh-performance liquid chromatography (HPLC) system (L-2000;manufactured by Hitachi High-Tech Science Corp.) connected with Superdex200 HR 10/30 (GE Healthcare Japan Corp.) column (eluent: 80 mM sodiumacetate buffer, pH 6+20% CH₃CN containing 80 mM Na₂SO₄, flow rate: 0.5mL/min). The preparative HPLC yielded 1.0 mg (based on h.AM (1-52)) ofthe compound (3) of interest.

Experiment I-1-4: Synthesis of CH₃O-PEG (20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52)) (Compound (4))

1 mg of the h.AM (1-52) peptide was dissolved in a 100 mM sodium acetatebuffer (pH 5.5) to obtain 1 mL of a peptide solution. To this peptidesolution, 32 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-2)(CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecular weightof 20 kDa was added under ice cooling. To this peptide solution, NaCNBH₃was further added so as to attain a final concentration of 20 mM. Thereaction solution was left at 4° C. for 24 hours. The obtained reactionsolution was diluted 5-fold with a 50 mM sodium acetate buffer (pH 4.0).The diluted reaction solution was applied at a flow rate of 2 mL/hr toSP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL) equilibratedwith a 50 mM sodium acetate buffer (pH 4.0). The column was washed with2 mL of a 50 mM sodium acetate buffer (pH 4.0). Subsequently, 5 mL of a50 mM sodium acetate buffer (pH 5.0) containing 1 M NaCl was applied tothe column to obtain an eluted fraction. An alkylamine linkage-type PEG(20k) adrenomedullin derivative (CH₃O-PEG (20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52))) (4) and unreacted h.AM (1-52) peptide were recovered in theeluted fraction. This eluted fraction was concentrated into 0.2 mL usingan ultrafiltration membrane (Amicon Ultra 4, Merck Millipore). Theobtained concentrate was purified and fractionated using a HPLC system(L-2000; manufactured by Hitachi High-Tech Science Corp.) connected withSuperdex 200 HR 10/30 (GE Healthcare Japan Corp.) column (eluent: 80 mMsodium acetate buffer, pH 6+20% CH₃CN containing 80 mM Na₂SO₄, flowrate: 0.5 mL/min). The preparative HPLC yielded 0.3 mg (based on h.AM(1-52)) of the compound (4) of interest.

Experiment I-1-5: Synthesis of CH₃O-PEG (30k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52)) (Compound (5))

2 mg of the h.AM (1-52) peptide was dissolved in a 100 mM sodium acetatebuffer (pH 5.5) to obtain 2 mL of a peptide solution. To this peptidesolution, 30 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-2)(CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecular weightof 30 kDa was added under ice cooling. To this peptide solution, NaCNBH₃was further added so as to attain a final concentration of 20 mM. Thereaction solution was left at 4° C. for 24 hours. The obtained reactionsolution was diluted 5-fold with a 50 mM sodium acetate buffer (pH 4.0).The diluted reaction solution was applied at a flow rate of 2 mL/hr toSP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL) equilibratedwith a 50 mM sodium acetate buffer (pH 4.0). The column was washed with2 mL of a 50 mM sodium acetate buffer (pH 4.0). Subsequently, 5 mL of a50 mM sodium acetate buffer (pH 5.0) containing 1 M NaCl was applied tothe column to obtain an eluted fraction. An alkylamine linkage-type PEG(30k) adrenomedullin derivative (CH₃O-PEG (30k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52))) (5) and unreacted h.AM (1-52) peptide were recovered in theeluted fraction. This eluted fraction was concentrated into 0.2 mL usingan ultrafiltration membrane (Amicon Ultra 4, Merck Millipore). Theobtained concentrate was purified and fractionated using a HPLC system(L-2000; manufactured by Hitachi High-Tech Science Corp.) connected withSuperdex 200 HR 10/30 (GE Healthcare Japan Corp.) column (eluent: 100 mMsodium acetate buffer, pH 6+200 mM Na₂SO₄, flow rate: 0.5 mL/min). Thepreparative HPLC yielded 0.8 mg (based on h.AM (1-52)) of the compound(5) of interest.

Experiment I-1-6: Synthesis of GL-2-Branched CH₃O-PEG(20k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (6))

The same procedures as in experiment I-1-5 were carried out except that45 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 20 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(20k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(20k)-CH₂—^(α)NH-(h.AM (1-52))) (6):

Preparative HPLC yielded 1.0 mg (based on h.AM (1-52)) of the compound(6) of interest.

Experiment I-1-7: Synthesis of GL-2-Branched CH₃O-PEG (40k)-CH₂—NH-(h.AM(1-52)) (Compound (7))

The same procedures as in experiment I-1-5 were carried out except that80 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 40 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(40k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52))) (7):

Preparative HPLC yielded 1.2 mg (based on h.AM (1-52)) of the compound(7) of interest.

Experiment I-1-8: Preparation of GL-2-Branched CH₃O-PEG(60k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (8))

The same procedures as in experiment I-1-4 were carried out except that40 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 60 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(60k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(60k)-CH₂—NH-(h.AM (1-52))) (8):

Preparative HPLC yielded 0.4 mg (based on h.AM (1-52)) of the compound(8) of interest.

Experiment I-1-9: Synthesis of GL-2-Branched CH₃O-PEG(80k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (9))

The same procedures as in experiment I-1-5 were carried out except that121 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 80 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(80k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(60k)-CH₂—^(α)NH-(h.AM (1-52))) (9):

Preparative HPLC yielded 1.1 mg (based on h.AM (1-52)) of the compound(9) of interest.

Experiment I-1-10: Synthesis of Lys-2-Branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (10))

The same procedures as in experiment I-1-4 were carried out except that42.9 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-4) with aweight-average molecular weight of 40 kDa represented by formula(VI-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain alysine skeleton-containing 2-branched alkylamine linkage-type PEG (40k)adrenomedullin derivative (Lys-2-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52))) (10):

Preparative HPLC yielded 0.4 mg (based on h.AM (1-52)) of the compound(10) of interest.

Experiment I-1-11: Synthesis of GL-4-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (11))

The same procedures as in experiment I-1-3 were carried out except that93 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-5) with aweight-average molecular weight of 40 kDa represented by formula(VII-2-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 4-branched alkylamine linkage-type PEG(40k) adrenomedullin derivative (GL-4-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52))) (11):

Preparative HPLC yielded 1.1 mg (based on h.AM (1-52)) of the compound(11) of interest.

Experiment I-1-12: Synthesis of Xyl-4-Branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (12))

The same procedures as in experiment I-1-3 were carried out except that94 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-6) with aweight-average molecular weight of 40 kDa represented by formula(VIII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain axylose skeleton-containing 4-branched alkylamine linkage-type PEG (40k)adrenomedullin derivative (Xyl-4-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (1-52))) (12):

Preparative HPLC yielded 1.0 mg (based on h.AM (1-52)) of the compound(12) of interest.

Experiment I-1-13: Synthesis of GL-3-branched CH₃O-PEG(50k)-CH₂—^(α)NH-(h.AM (1-52)) (Compound (13))

The same procedures as in experiment I-1-3 were carried out except that94 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-7) with aweight-average molecular weight of 50 kDa represented by formula(VII-1-2′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 3-branched alkylamine linkage-type PEG(50k) adrenomedullin derivative (GL-3-branched CH₃O-PEG(50k)-CH₂—^(α)NH-(h.AM (1-52))) (13):

Preparative HPLC yielded 0.9 mg (based on h.AM (1-52)) of the compound(13) of interest.

Experiment I-1-14: Synthesis of CH₃O-PEG (20k)-CO—^(α)NH-(h.AM (1-52))(Compound (14))

A Cys¹⁶-Cys²¹ disulfide bridge form of a peptide having the amino acidsequence ofFmoc-Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “Fmoc-^(α)NH-(h.AM (1-52))”) wassynthesized on commission by the Fmoc peptide synthesis method. 18 mg ofthe Fmoc-^(α)NH-(h.AM (1-52)) peptide was dissolved in 1.8 mL ofdimethyl sulfoxide (DMSO). To this solution, 9 mg of t-butylsuccinimidyl carbonate and 6 μL of diisopropylethylamine were added. Thereaction solution was stirred for 5 hours. To the obtained reactionsolution, an aqueous acetic acid solution was added. Then, this solutionwas freeze-dried. The residue was dissolved in 2 mL of DMSO. To theobtained solution, 0.2 mL of diethylamine was added. The obtainedsolution was stirred for 70 minutes. The reaction solution was dilutedby the addition of an aqueous acetic acid solution. The obtainedsolution was fractionated using reverse-phase HPLC to obtain a fractioncontaining the h.AM (1-52) peptide. This fraction was freeze-dried toobtain 10 mg of h.AM (1-52) peptide having 4 lysine residues protectedwith Boc groups as a white powder.

2 mg of the obtained peptide was dissolved in 2 mL of DMSO. To thispeptide solution, 15 mg of a p-nitrophenyl ester-type CH₃O-PEGylationreagent (PEG-8) (CH₃O—(CH₂CH₂O)_(n)—CO—O—C₆H₄-p-NO₂) with aweight-average molecular weight of 20 kDa was added under ice cooling.To this peptide solution, 6.5 μL of a 0.1 M solution of triethylamine inDMSO was further added. The reaction solution was left for 1 hour underice cooling. Then, the reaction solution was brought back to roomtemperature and left for 24 hours. The temperature of the reactionsolution was further raised to 30° C., and the reaction was continuedfor 2 days. The reaction solution was freeze-dried. To the obtainedresidue, 1 mL of trifluoroacetic acid was added under ice cooling. Themixture was brought back to room temperature and left for 2 hours.Subsequently, the trifluoroacetic acid was distilled away under reducedpressure from the mixture using an evaporator. The obtained residue wasdissolved by the addition of 4 mL of a 50 mM sodium acetate buffer (pH4.0). This solution was applied at a flow rate of 1 mL/hr toSP-Sepharose HP (GE Healthcare Japan Corp.) column (1 mL) equilibratedwith a 50 mM sodium acetate buffer (pH 4.0). The column was washed with2 mL of a 50 mM sodium acetate buffer (pH 4.0). Subsequently, 5 mL of a50 mM sodium acetate buffer (pH 5.0) containing 1 M NaCl was applied tothe column to obtain an eluted fraction. A urethane linkage-type PEG(20k) adrenomedullin derivative (CH₃O-PEG (20k)-CO—^(α)NH-(h.AM (1-52)))(14) and unreacted h.AM (1-52) peptide were recovered in the elutedfraction. This eluted fraction was concentrated into 0.2 mL using anultrafiltration membrane (Amicon Ultra 4, Merck Millipore). The obtainedconcentrate was purified and fractionated using a HPLC system (L-2000;manufactured by Hitachi High-Tech Science Corp.) connected with Tsk gelG2000SWxL (60 cm, Tosoh Corp.) column (eluent: 80 mM sodium acetatebuffer, pH 6+20% CH₃CN containing 80 mM Na₂SO₄, flow rate: 0.5 mL/min).The preparative HPLC yielded 250 μg (based on h.AM (1-52)) of thecompound (14) of interest.

Experiment I-1-15: Synthesis of CH₃O-PEG (5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52)) (Compound (35))

The same procedures as in experiment I-1-3 were carried out except that5 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-2)(CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecular weightof 5 kDa was used instead of the aldehyde-type CH₃O-PEGylation reagent(PEG-2) with a weight-average molecular weight of 10 kDa, to obtain analkylamine linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₂—CH₂-^(α)NH-(h.AM (1-52))) (35). Preparative HPLC yielded 0.8mg (based on h.AM (1-52)) of the compound (35) of interest.

Experiment I-1-16: Synthesis of CH₃O-PEG (40k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(1-52)) (Compound (25))

The same procedures as in experiment I-1-3 were carried out except that40 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-2)(CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecular weightof 40 kDa was used instead of the aldehyde-type CH₃O-PEGylation reagent(PEG-2) with a weight-average molecular weight of 10 kDa, to obtain analkylamine linkage-type PEG (40k) adrenomedullin derivative (CH₃O-PEG(40k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (1-52))) (25). Preparative HPLC yielded0.6 mg (based on h.AM (1-52)) of the compound (25) of interest.

Experiment I-1-17: Synthesis of GL-2-Branched CH₃O-PEG(20k)-CO—^(α)NH-(h.AM (1-52)) (Compound (26))

The same procedures as in experiment I-1-14 were carried out except that25 mg of a p-nitrophenyl ester-type CH₃O-PEGylation reagent (PEG-9) witha weight-average molecular weight of 20 kDa represented by formula(XII-1-1′):

was used instead of the p-nitrophenyl ester-type CH₃O-PEGylation reagent(PEG-8) (CH₃O—(CH₂CH₂O)_(n)—CO—O—C₆H₄-p-NO₂) with a weight-averagemolecular weight of 20 kDa, to obtain a glycerol skeleton-containing2-branched urethane linkage-type PEG (20k) adrenomedullin derivative(GL-2-branched CH₃O-PEG (20k)-CO—^(α)NH-(h.AM (1-52)) (26):

Preparative HPLC yielded 0.2 mg (based on h.AM (1-52)) of the compound(26) of interest.

Experiment I-1-18: Synthesis of GL-2-Branched CH₃O-PEG(40k)-CO—^(α)NH-(h.AM (1-52)) (Compound (27))

The same procedures as in experiment I-1-14 were carried out except that35 mg of a p-nitrophenyl ester-type CH₃O-PEGylation reagent (PEG-9) witha weight-average molecular weight of 40 kDa represented by formula(XII-1-1′):

was used instead of the p-nitrophenyl ester-type CH₃O-PEGylation reagent(PEG-8) (CH₃O—(CH₂CH₂O)_(n)—CO—O—C₆H₄-p-NO₂) with a weight-averagemolecular weight of 20 kDa, to obtain a glycerol skeleton-containing2-branched urethane linkage-type PEG (40k) adrenomedullin derivative(GL-2-branched CH₃O-PEG (40k)-CO—^(α)NH-(h.AM (1-52)) (27):

Preparative HPLC yielded 0.2 mg (based on h.AM (1-52)) of the compound(27) of interest.

Experiment I-1-19: Synthesis of GL-4-branched CH₃O-PEG(40k)-CO—^(α)NH-(h.AM (1-52)) (Compound (28))

The same procedures as in experiment I-1-14 were carried out except that40 mg of a p-nitrophenyl ester-type CH₃O-PEGylation reagent (PEG-10)with a weight-average molecular weight of 40 kDa represented by formula(XII-2-1′):

was used instead of the p-nitrophenyl ester-type CH₃O-PEGylation reagent(PEG-8) (CH₃O—(CH₂CH₂O)_(n)—CO—O—C₆H₄-p-NO₂) with a weight-averagemolecular weight of 20 kDa, to obtain a glycerol skeleton-containing4-branched urethane linkage-type PEG (40k) adrenomedullin derivative(GL-4-branched CH₃O-PEG (40k)-CO—^(α)NH-(h.AM (1-52)) (28):

Preparative HPLC yielded 0.2 mg (based on h.AM (1-52)) of the compound(28) of interest.

Experiment I-2: Structural Analysis of Full-Length AdrenomedullinDerivative Experiment I-2-1: Identification of Binding Position to PEGGroup by Mass Spectrometry of Cleaved Peptide—(1)

10 μg of the compound (3) was mixed with 70% formic acid and 600 μg ofcyanogen bromide (BrCN) to adjust the total amount to 500 μL. Thismixture was reacted overnight at room temperature. 1 mL each ofchloroform, methanol, and a 60% aqueous acetonitrile solution containing0.1% trifluoroacetic acid were applied, in order, to Sep Pak (WatersCorp.) column, and the column was washed. Then, the Sep Pak column wasequilibrated by the application of 1 mL of ultrapure water. To thecyanogen bromide-treated reaction solution (500 μL) reacted overnight,4,500 μL of ultrapure water was added to obtain 5 mL of a dilutedreaction solution. The diluted reaction solution was applied to thecolumn for adsorption of a cleaved peptide. Then, 1 mL each of ultrapurewater and a 10% aqueous acetonitrile solution containing 0.1%trifluoroacetic acid were applied, in order, to the column, and thecolumn was washed to remove unadsorbed substances. Finally, 1 mL of a60% aqueous acetonitrile solution containing 0.1% trifluoroacetic acidwas applied to the column to elute the cleaved peptide from the column.

The acetonitrile was distilled off under reduced pressure from theeluted fraction of the cleaved peptide thus obtained from the Sep Pakcolumn. The obtained residue was purified and fractionated byreverse-phase HPLC (RP-HPLC) using a reverse-phase column (ODS-120ATSKgel, Tosoh Corp.). The elution in RP-HPLC was carried out under alinear gradient program of changing from 100% solution A (10% aqueousacetonitrile solution containing 0.1% trifluoroacetic acid) to 100%solution B (60% aqueous acetonitrile solution containing 0.1%trifluoroacetic acid) over 60 minutes. The MS spectrum of the separatedcleaved peptide was measured using a mass spectrometry apparatus(AXIMA-confidence, Shimadzu Corp.). As a result, the molecular weight ofthe cleaved peptide was confirmed to agree with the molecular weight ofa peptide corresponding to amino acid residues 6 to 52 of humanadrenomedullin ((M+Na)⁺, calcd: m/z 5385.935; found: m/z 5385.9986). Alllysine residues (residues 25, 36, 38 and 46 from the N terminus) presentin human adrenomedullin are located within the range from amino acidresidues 6 to 52. From these results, therefore, the PEG group in thealkylamine linkage-type PEG (10k) adrenomedullin derivative (3) wasconfirmed to be attached to the N-terminal α-amino group.

Experiment I-2-2: Identification of Binding Position to PEG Group byMass Spectrometry of Cleaved Peptide—(2)

10 to 40 μg of the compound (2) was dissolved in 500 μL of a solutioncontaining 2.5 mM ethylenediaminetetraacetic acid, 30%N,N-dimethylformamide and 250 mM Tris-HCl (pH 8.5), and the solution wasstirred and mixed by a vortex mixer and ultrasonication. To the mixture,2.5 mg of 1,4-dithiothreitol was added, and the resulting mixture wasconfirmed to have pH of 8.0 or higher. To the mixture, nitrogen gas wasinjected, followed by ultrasonication for 5 minutes. This mixture wasreacted at 37° C. for 2 hours. To the reaction mixture thus reacted,6.25 mg of monoiodoacetic acid was added in the dark, and the mixturewas further reacted at 25° C. for 30 minutes. Then, the reaction wasterminated by the addition of acetic acid (final concentration: 1 N) tothe reaction mixture. 1 mL each of chloroform, methanol, and a 60%aqueous acetonitrile solution containing 0.1% trifluoroacetic acid wereapplied, in order, to Sep Pak (Waters Corp.) column, and the column waswashed. Then, the Sep Pak column was equilibrated by the application of1 mL of 1 N acetic acid. The reductively alkylated reaction solution wasapplied to the column for adsorption of a reacted peptide. Then, 1 mLeach of 1 N acetic acid and a 10% aqueous acetonitrile solutioncontaining 0.1% trifluoroacetic acid were applied, in order, to thecolumn, and the column was washed to remove unadsorbed substances.Finally, 1 mL of a 60% aqueous acetonitrile solution containing 0.1%trifluoroacetic acid was applied to the column to elute the reductivelyalkylated peptide from the column.

The acetonitrile was distilled off under reduced pressure from theeluted fraction of the reductively alkylated peptide thus obtained fromthe Sep Pak column. The obtained reductively alkylated peptide was mixedwith lysyl endopeptidase at a peptide:lysyl endopeptidase mass ratio of20:1. To the mixture, 1 M Tris-HCl (pH 8.5) was added to adjust thevolume to 200 μL and the final concentration of Tris-HCl to 50 mM. Thismixture was left overnight (16 hours or longer) at 37° C. The obtainedcleaved peptide was purified and separated by RP-HPLC using areverse-phase column (ODS-120A TSKgel, Tosoh Corp.). The elution inRP-HPLC was carried out under a program of applying 100% solution A(0.1% trifluoroacetic acid) for 5 minutes, then applying an eluent underlinear gradient conditions involving changing from the 100% solution Ato 50% solution B (60% aqueous acetonitrile solution containing 0.1%trifluoroacetic acid) over 60 minutes, and further applying 100%solution B for 15 minutes. Reactions and preparative RP-HPLC werecarried out by the same procedures as above using a chemicallysynthesized h.AM (1-52) peptide preparation as a control instead of thecompound (2). FIG. 1 shows the RP-HPLC chromatogram of the cleavedpeptide. FIG. 1A shows the RP-HPLC chromatogram of the cleaved peptidederived from the h.AM (1-52) peptide, and FIG. 1B shows the RP-HPLCchromatogram of the cleaved peptide derived from the compound (2). Asshown in FIG. 1A, 4 major peaks were detected at retention times of28.08 minutes (hereinafter, also described as “peak (1)”), 36.97 minutes(hereinafter, also described as “peak (2)”), 54.53 minutes (hereinafter,also described as “peak (3)”), and 67.52 minutes (hereinafter, alsodescribed as “peak (4)”) in the RP-HPLC chromatogram of the cleavedpeptide derived from the h.AM (1-52) peptide. On the other hand, asshown in FIG. 1B, 4 major peaks were detected at retention times of28.69 minutes (hereinafter, also described as “peak (5)”), 36.98 minutes(hereinafter, also described as “peak (6)”), 54.57 minutes (hereinafter,also described as “peak (7)”), and 72.30 minutes (hereinafter, alsodescribed as “peak (8)”) in the RP-HPLC chromatogram of the cleavedpeptide derived from the compound (2). From the comparison of theretention times, the peaks (1) and (5), the peaks (2) and (6), or thepeaks (3) and (7) correspond to the same peptide fragment. The peaks (4)and (8) differ in retention time. The compound of the peak (8) ispresumed to be a compound having a PEG group attached to the peptidefragment of the peak (4).

Reactions and preparative RP-HPLC were carried out by the sameprocedures as above using the compounds (7), (8) and (26). As a result,peaks having retention times corresponding to the peaks (1), (2) and (3)were detected, as in the case of using the compound (2). Also, a peakpresumed to correspond to a compound having a PEG group attached to thepeptide fragment of the peak (4) was detected, as in the peak (8).

The MS spectrum of the separated cleaved peptide was measured using amass spectrometry apparatus (QSTAR Elite, AB Sciex Pte. Ltd). Humanadrenomedullin has 4 lysine residues (residues 25, 36, 38 and 46 fromthe N terminus). Therefore, the cleaved peptide obtained by lysylendopeptidase consists of 5 peptide fragments, specifically, peptidefragments of YRQSMNNFQGLRSFGCRFGTCTVQK (h.AM (1-25)), LAHQIYQFTAK (h.AM(26-36)), DK (h.AM (37-38)), DNVAPRSK (h.AM (39-46)), and ISPQGY (h.AM(47-52)) from the N terminus. From the obtained MS spectrum, the peaks(1) and (5) were confirmed to correspond to the peptide fragment of h.AM(39-46), the peaks (2) and (6) were confirmed to correspond to thepeptide fragment of h.AM (47-52), the peaks (3) and (7) were confirmedto correspond to the peptide fragment of h.AM (26-36), and the peak (4)was confirmed to correspond to the peptide fragment of h.AM (1-25). As aresult of measuring the MS spectrum of the peptide fragment of the peak(8) using a mass spectrometry apparatus (autoflex II, Bruker DaltonicsK.K.), the compound of the peak (8) was confirmed to be a compoundhaving a PEG group attached to the N-terminal peptide fragment of theh.AM (1-52) peptide. From these results, therefore, all the PEG groupsin the compounds (7), (8) and (26) were confirmed to be attached to theN-terminal α-amino groups.

Experiment I-2-3: Identification of Binding Position to PEG Group byAmino Acid Sequence Analysis

The compounds (3), (4), (5), (6), (7), (8), (9), (10), (11), (12) and(13) were subjected to amino acid sequence analysis using a proteinsequencer (Procise 494 HT Protein Sequencing System, Applied Biosystems,Inc.). As a result, an amino acid corresponding to the N-terminal aminoacid residue of human adrenomedullin was detected in none of thecompounds. From these results, all the PEG groups in the compounds (3),(4), (5), (6), (7), (8), (9), (10), (11), (12) and (13) were confirmedto be attached to the N-terminal α-amino groups.

Experiment I-2-4: Identification of Binding Position to PEG Group byIon-Exchange HPLC

The peptide corresponding to amino acid residues 6 to 52 of humanadrenomedullin was separated from the h.AM (1-52) peptide byion-exchange HPLC using an ion-exchange column (CM-2SW, Tosoh Corp.).The elution in ion-exchange HPLC was carried out under a linear gradientprogram of changing from 80% solution A (100 mM sodium acetate, pH 5.0)and 20% solution B (100 mM sodium acetate containing 1 M sodium sulfate,pH 7.0) to 20% solution A and 80% solution B from 0 to 40 minutes.

The acetonitrile was distilled off under reduced pressure from theeluted fraction of the cleaved peptide of the compound (3) obtained fromthe Sep Pak column in experiment I-2-1. The residue was analyzed byion-exchange HPLC under the conditions described above. As a result, apeak having the same elution time as that of the peptide correspondingto amino acid residues 6 to 52 of human adrenomedullin was confirmed asto the compound (3). The acetonitrile was distilled off under reducedpressure from the eluted fractions of the cleaved peptides of thecompounds (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (25),(27), (28) and (35) obtained by the same procedures as in experimentI-2-1 from the Sep Pak column. As a result of analyzing the residues byion-exchange HPLC under the conditions described above, their peaks wereconfirmed to agree with the peak of the compound (3). From theseresults, therefore, all the PEG groups in the compounds (3), (4), (5),(6), (7), (8), (9), (10), (11), (12), (13), (25), (27), (28) and (35)were confirmed to be attached to the N-terminal α-amino groups.

Experiment I-2-5: Molecular Weight Analysis by SDS-PAGE

In accordance with the laboratory textbook (Experimental Medicine,Suppl., “Handbook for Protein Experiments”, Yodosha Co., Ltd., edited byTadaomi Takenawa and Toshiki Ito), the compounds (1), (2), (3), (4),(5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (25), (26), (27),(28) and (35) (200 ng each) obtained in experiment I-1 were separated bySDS-PAGE using a polyacrylamide gel with a concentration gradient from10% to 20%. The results are shown in FIGS. 2, 3 and 4. In FIG. 2, lane 0depicts a molecular weight standard, lane 1 depicts the compound (3),lane 2 depicts the compound (4), lane 3 depicts the compound (5), lane 4depicts the compound (6), lane 5 depicts the compound (7), lane 6depicts the compound (8), lane 7 depicts the compound (9), lane 8depicts the compound (10), lane 9 depicts the compound (11), and lane 10depicts the compound (12). In FIG. 3, lane 0 depicts a molecular weightstandard, lane 1 depicts the compound (1), lane 2 depicts the compound(2), lane 3 depicts the compound (13), lane 4 depicts the compound (14),lane 5 depicts compound (15) mentioned later, lane 6 depicts compound(16) mentioned later, and lane 7 depicts compound (17) mentioned later.In FIG. 4, lane 0 depicts a molecular weight standard, lane 1 depictsthe compound (25), lane 2 depicts the compound (26), lane 3 depicts thecompound (27), lane 4 depicts the compound (28), lane 5 depicts compound(29) mentioned later, lane 6 depicts compound (30) mentioned later, lane7 depicts compound (31) mentioned later, lane 8 depicts compound (32)mentioned later, lane 9 depicts compound (33) mentioned later, lane 10depicts compound (34) mentioned later, lane 11 depicts the compound(35), lane 12 depicts compound (36) mentioned later, and lane 13 depictscompound (37) mentioned later. All the molecular weight standards usedwere Precision Plus Protein™ Dual Xtra Standards (Bio-Rad Laboratories,Inc.). As shown in FIGS. 2, 3 and 4, each compound was confirmed to havethe desired molecular weight.

Experiment I-2-6: Confirmation of Association by Gel Filtration HPLC

Association of adrenomedullin derivative molecules was confirmed by gelfiltration HPLC using a gel filtration column (Superdex 200 Increase10/300 GL, GE Healthcare Japan Corp.). The compounds (3), (4), (5), (6),(7), (8), (9), (10), (11), (12), (13), (25), (27), (28), and (35) (50 μgeach) obtained in experiment I-1 were added to the column. An eluent(100 mM sodium acetate and 100 mM sodium sulfate, pH 6.0) was applied tothe column at a flow rate of 0.75 mL/min. From the obtained gelfiltration chromatogram, each compound exhibited a single peak having aretention time appropriate for the molecular weight. From these results,each adrenomedullin derivative molecule was confirmed to be present as amonomer without being associated. The retention time of each compound inthe gel filtration chromatogram is shown in Table 1.

TABLE 1 Adrenomedullin derivative Retention time (min) Compound (3) 18.6Compound (4) 16.2 Compound (5) 14.6 Compound (6) 16.4 Compound (7) 13.7Compound (8) 12.9 Compound (9) 11.9 Compound (10) 13.8 Compound (11)14.1 Compound (12) 14.4 Compound (13) 13.3 Compound (25) 13.9 Compound(27) 13.9 Compound (28) 14.5 Compound (35) 21.2

Experiment II: Preparation of N-Terminally Deleted AdrenomedullinDerivative Experiment II-1: Synthesis of N-Terminally DeletedAdrenomedullin Derivative Experiment II-1-1: Synthesis of CH₃O-PEG(5k)-(CH₂)₅—CO—^(α)NH-(h.AM (6-52)) (Compound (15))

In accordance with the method described in the known literature (Kubo, Ket al., “Biological properties of adrenomedullin conjugated withpolyethylene glycol.”, Peptides, 2014, vol. 57, p. 118-21), apolyethylene glycol group with a weight-average molecular weight of 5kDa (PEG (5k)) was linked via an amide bond to the N-terminal aminogroup of a Cys¹⁶-Cys²¹ disulfide bridge form of a peptide having theamino acid sequence ofH-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “h.AM (6-52)”), which was a peptidecorresponding to amino acid residues 6 to 52 of human adrenomedullin,using a N-hydroxysuccinimide active ester-type CH₃O-PEGylation reagent(PEG-1) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₅—CO—O—NHS) of 5 kDa to synthesize anamide linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₅—CO—^(α)NH-(h.AM (6-52))) (15).

Experiment II-1-2: Synthesis of CH₃O-PEG (5k)-(CH₂)₅—CO—^(α)NH-(h.AM(11-52)) (Compound (16))

In the same way as in experiment II-1-1, a polyethylene glycol groupwith a weight-average molecular weight of 5 kDa (PEG (5k)) was linkedvia an amide bond to the N-terminal amino group of a Cys¹⁶-Cys²¹disulfide bridge form of a peptide having the amino acid sequence ofH-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “h.AM (11-52)”), which was a peptidecorresponding to amino acid residues 11 to 52 of human adrenomedullin,using a N-hydroxysuccinimide active ester-type CH₃O-PEGylation reagent(PEG-1) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₅—CO—O—NHS) of 5 kDa to synthesize anamide linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₅—CO—^(α)NH-(h.AM (11-52))) (16).

Experiment II-1-3: Synthesis of CH₃O-PEG (5k)-(CH₂)₅—CO—^(α)NH-(h.AM(16-52)) (Compound (17))

In the same way as in experiment II-1-1, a polyethylene glycol groupwith a weight-average molecular weight of 5 kDa (PEG (5k)) was linkedvia an amide bond to the N-terminal amino group of a Cys¹⁶-Cys²¹disulfide bridge form of a peptide having the amino acid sequence ofH-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “h.AM (16-52)”), which was a peptidecorresponding to amino acid residues 16 to 52 of human adrenomedullin,using a N-hydroxysuccinimide active ester-type CH₃O-PEGylation reagent(PEG-1) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₅—CO—O—NHS) of 5 kDa to synthesize anamide linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₅—CO—^(α)NH-(h.AM (16-52))) (17).

Experiment II-1-4: Synthesis of CH₃O-PEG (5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(6-52)) (Compound (18))

0.4 mg of the h.AM (6-52) peptide was dissolved in a 100 mM sodiumacetate buffer (pH 5.5) to obtain 0.5 mL of a peptide solution. To thispeptide solution, 2 mg of an aldehyde-type CH₃O-PEGylation reagent(PEG-2) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecularweight of 5 kDa was added under ice cooling. To this peptide solution,NaCNBH₃ was further added so as to attain a final concentration of 20mM. The reaction solution was left at 4° C. for 24 hours. The obtainedreaction solution was diluted 5-fold with a 50 mM sodium acetate buffer(pH 4.0). The diluted reaction solution was applied at a flow rate of 2mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL)equilibrated with a 50 mM sodium acetate buffer (pH 4.0). The column waswashed with 2 mL of a 50 mM sodium acetate buffer (pH 4.0).Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0) containing1 M NaCl was applied to the column to obtain an eluted fraction. Analkylamine linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (6-52))) (18) and unreacted h.AM (6-52)peptide were recovered in the eluted fraction. This eluted fraction wasconcentrated into 0.2 mL using an ultrafiltration membrane (Amicon Ultra4, Merck Millipore). The obtained concentrate was purified andfractionated using a HPLC system (L-2000; manufactured by HitachiHigh-Tech Science Corp.) connected with Tsk gel G2000SWxL (60 cm, TosohCorp.) column (eluent: 80 mM sodium acetate buffer, pH 6+20% CH₃CNcontaining 80 mM Na₂SO₄, flow rate: 0.5 mL/min). The preparative HPLCyielded 0.12 mg (based on h.AM (6-52)) of the compound (18) of interest.

Experiment II-1-5: Synthesis of CH₃O-PEG (5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(11-52)) (Compound (19))

0.44 mg of the h.AM (11-52) peptide was dissolved in a 100 mM sodiumacetate buffer (pH 5.5) to obtain 0.5 mL of a peptide solution. To thispeptide solution, 2.5 mg of an aldehyde-type CH₃O-PEGylation reagent(PEG-2) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecularweight of 5 kDa was added under ice cooling. To this peptide solution,NaCNBH₃ was further added so as to attain a final concentration of 20mM. The reaction solution was left at 4° C. for 24 hours. The obtainedreaction solution was diluted 5-fold with a 50 mM sodium acetate buffer(pH 4.0). The diluted reaction solution was applied at a flow rate of 2mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL)equilibrated with a 50 mM sodium acetate buffer (pH 4.0). The column waswashed with 2 mL of a 50 mM sodium acetate buffer (pH 4.0).Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0) containing1 M NaCl was applied to the column to obtain an eluted fraction. Analkylamine linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (11-52))) (19) and unreacted h.AM (11-52)peptide were recovered in the eluted fraction. This eluted fraction wasconcentrated into 0.2 mL using an ultrafiltration membrane (Amicon Ultra4, Merck Millipore). The obtained concentrate was purified andfractionated using a HPLC system (L-2000; manufactured by HitachiHigh-Tech Science Corp.) connected with Tsk gel G2000SWxL (60 cm, TosohCorp.) column (eluent: 80 mM sodium acetate buffer, pH 6+20% CH₃CNcontaining 80 mM Na₂SO₄, flow rate: 0.5 mL/min). The preparative HPLCyielded 0.1 mg (based on h.AM (11-52)) of the compound (19) of interest.

Experiment II-1-6: Synthesis of CH₃O-PEG (5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(16-52)) (Compound (20))

0.46 mg of the h.AM (16-52) peptide was dissolved in a 100 mM sodiumacetate buffer (pH 5.5) to obtain 0.5 mL of a peptide solution. To thispeptide solution, 3 mg of an aldehyde-type CH₃O-PEGylation reagent(PEG-2) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecularweight of 5 kDa was added under ice cooling. To this peptide solution,NaCNBH₃ was further added so as to attain a final concentration of 20mM. The reaction solution was left at 4° C. for 24 hours. The obtainedreaction solution was diluted 5-fold with a 50 mM sodium acetate buffer(pH 4.0). The diluted reaction solution was applied at a flow rate of 2mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL)equilibrated with a 50 mM sodium acetate buffer (pH 4.0). The column waswashed with 2 mL of a 50 mM sodium acetate buffer (pH 4.0).Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0) containing1 M NaCl was applied to the column to obtain an eluted fraction. Analkylamine linkage-type PEG (5k) adrenomedullin derivative (CH₃O-PEG(5k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (16-52))) (20) and unreacted h.AM (16-52)peptide were recovered in the eluted fraction. This eluted fraction wasconcentrated into 0.2 mL using an ultrafiltration membrane (Amicon Ultra4, Merck Millipore). The obtained concentrate was purified andfractionated using a HPLC system (L-2000; manufactured by HitachiHigh-Tech Science Corp.) connected with Tsk gel G2000SWxL (60 cm, TosohCorp.) column (eluent: 80 mM sodium acetate buffer, pH 6+20% CH₃CNcontaining 80 mM Na₂SO₄, flow rate: 0.5 mL/min). The preparative HPLCyielded 0.15 mg (based on h.AM (16-52)) of the compound (20) ofinterest.

Experiment II-1-7: Synthesis of CH₃O-PEG (20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(6-52)) (Compound (21))

0.22 mg of the h.AM (6-52) peptide was dissolved in a 100 mM sodiumacetate buffer (pH 5.5) to obtain 0.2 mL of a peptide solution. To thispeptide solution, 4.1 mg of an aldehyde-type CH₃O-PEGylation reagent(PEG-2) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecularweight of 20 kDa was added under ice cooling. To this peptide solution,NaCNBH₃ was further added so as to attain a final concentration of 20mM. The reaction solution was left at 4° C. for 24 hours. The obtainedreaction solution was diluted 5-fold with a 50 mM sodium acetate buffer(pH 4.0). The diluted reaction solution was applied at a flow rate of 2mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL)equilibrated with a 50 mM sodium acetate buffer (pH 4.0). The column waswashed with 2 mL of a 50 mM sodium acetate buffer (pH 4.0).Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0) containing1 M NaCl was applied to the column to obtain an eluted fraction. Analkylamine linkage-type PEG (20k) adrenomedullin derivative (CH₃O-PEG(20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (6-52))) (21) and unreacted h.AM (6-52)peptide were recovered in the eluted fraction. This eluted fraction wasconcentrated into 0.2 mL using an ultrafiltration membrane (Amicon Ultra4, Merck Millipore). The obtained concentrate was purified andfractionated using a HPLC system (L-2000; manufactured by HitachiHigh-Tech Science Corp.) connected with Tsk gel G2000SWxL (60 cm, TosohCorp.) column (eluent: 80 mM sodium acetate buffer, pH 6+20% CH₃CNcontaining 80 mM Na₂SO₄, flow rate: 0.5 mL/min). The preparative HPLCyielded 0.1 mg (based on h.AM (6-52)) of the compound (21) of interest.

Experiment II-1-8: Synthesis of CH₃O-PEG (20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(11-52)) (Compound (22))

0.22 mg of the h.AM (11-52) peptide was dissolved in a 100 mM sodiumacetate buffer (pH 5.5) to obtain 0.2 mL of a peptide solution. To thispeptide solution, 4.6 mg of an aldehyde-type CH₃O-PEGylation reagent(PEG-2) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecularweight of 20 kDa was added under ice cooling. To this peptide solution,NaCNBH₃ was further added so as to attain a final concentration of 20mM. The reaction solution was left at 4° C. for 24 hours. The obtainedreaction solution was diluted 5-fold with a 50 mM sodium acetate buffer(pH 4.0). The diluted reaction solution was applied at a flow rate of 2mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL)equilibrated with a 50 mM sodium acetate buffer (pH 4.0). The column waswashed with 2 mL of a 50 mM sodium acetate buffer (pH 4.0).Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0) containing1 M NaCl was applied to the column to obtain an eluted fraction. Analkylamine linkage-type PEG (20k) adrenomedullin derivative (CH₃O-PEG(20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (11-52))) (22) and unreacted h.AM (11-52)peptide were recovered in the eluted fraction. This eluted fraction wasconcentrated into 0.2 mL using an ultrafiltration membrane (Amicon Ultra4, Merck Millipore). The obtained concentrate was purified andfractionated using a HPLC system (L-2000; manufactured by HitachiHigh-Tech Science Corp.) connected with Tsk gel G2000SWxL (60 cm, TosohCorp.) column (eluent: 80 mM sodium acetate buffer, pH 6+20% CH₃CNcontaining 80 mM Na₂SO₄, flow rate: 0.5 mL/min). The preparative HPLCyielded 0.1 mg (based on h.AM (11-52)) of the compound (22) of interest.

Experiment II-1-9: Synthesis of CH₃O-PEG (20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM(16-52)) (Compound (23))

0.22 mg of the h.AM (16-52) peptide was dissolved in a 100 mM sodiumacetate buffer (pH 5.5) to obtain 0.2 mL of a peptide solution. To thispeptide solution, 5.2 mg of an aldehyde-type CH₃O-PEGylation reagent(PEG-2) (CH₃O—(CH₂CH₂O)_(n)—(CH₂)₂—CHO) with a weight-average molecularweight of 20 kDa was added under ice cooling. To this peptide solution,NaCNBH₃ was further added so as to attain a final concentration of 20mM. The reaction solution was left at 4° C. for 24 hours. The obtainedreaction solution was diluted 5-fold with a 50 mM sodium acetate buffer(pH 4.0). The diluted reaction solution was applied at a flow rate of 2mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.) column (2 mL)equilibrated with a 50 mM sodium acetate buffer (pH 4.0). The column waswashed with 2 mL of a 50 mM sodium acetate buffer (pH 4.0).Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0) containing1 M NaCl was applied to the column to obtain an eluted fraction. Analkylamine linkage-type PEG (20k) adrenomedullin derivative (CH₃O-PEG(20k)-(CH₂)₂—CH₂—^(α)NH-(h.AM (16-52))) (23) and unreacted h.AM (16-52)peptide were recovered in the eluted fraction. This eluted fraction wasconcentrated into 0.2 mL using an ultrafiltration membrane (Amicon Ultra4, Merck Millipore). The obtained concentrate was purified andfractionated using a HPLC system (L-2000; manufactured by HitachiHigh-Tech Science Corp.) connected with Tsk gel G2000SWxL (60 cm, TosohCorp.) column (eluent: 80 mM sodium acetate buffer, pH 6+20% CH₃CNcontaining 80 mM Na₂SO₄, flow rate: 0.5 mL/min). The preparative HPLCyielded 0.1 mg (based on h.AM (16-52)) of the compound (23) of interest.

Experiment II-1-10: Synthesis of GL-2-Branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (16-52)) (Compound (24))

The same procedures as in experiment II-1-6 were carried out except that20 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 40 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(40k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (16-52))) (24):

Preparative HPLC yielded 0.2 mg (based on h.AM (16-52)) of the compound(24) of interest.

Experiment II-1-11: Synthesis of GL-2-Branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (6-52)) (Compound (29))

The same procedures as in experiment II-1-7 were carried out except that20 mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 40 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(40k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(40k)-CH₂—^(α)NH-(h.AM (6-52))) (29):

Preparative HPLC yielded 0.15 mg (based on h.AM (6-52)) of the compound(29) of interest.

Experiment II-1-12: Synthesis of GL-2-Branched CH₃O-PEG(20k)-CO—^(α)NH-(h.AM (6-52)) (Compound (30))

A Cys¹⁶-Cys²¹ disulfide bridge form of a peptide having the amino acidsequence ofFmoc-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “Fmoc-^(α)NH-(h.AM (6-52))”) wassynthesized by the Fmoc peptide synthesis method. 17 mg of theFmoc-^(α)NH-(h.AM (6-52)) peptide was dissolved in 1.8 mL of DMSO. Tothis solution, 9 mg of t-butyl succinimidyl carbonate and 6 μL ofdiisopropylethylamine were added. The reaction solution was stirred for5 hours. To the obtained reaction solution, an aqueous acetic acidsolution was added. Then, this solution was freeze-dried. The residuewas dissolved in 2 mL of DMSO. To the obtained solution, 0.2 mL ofdiethylamine was added. The obtained solution was stirred for 70minutes. The reaction solution was diluted by the addition of an aqueousacetic acid solution. The obtained solution was fractionated usingreverse-phase HPLC to obtain a fraction containing the h.AM (6-52)peptide. This fraction was freeze-dried to obtain 9 mg of h.AM (6-52)peptide having 4 lysine residues protected with Boc groups as a whitepowder.

2 mg of the obtained peptide was dissolved in 2 mL of DMSO. To thispeptide solution, 15 mg of a p-nitrophenyl ester-type GL2-branchedCH₃O-PEGylation reagent (PEG-9) with a weight-average molecular weightof 20 kDa represented by formula (XII-1-1′):

was added under ice cooling. To this peptide solution, 6.5 μL of a 0.1 Msolution of triethylamine in DMSO was further added. The reactionsolution was left for 1 hour under ice cooling. Then, the reactionsolution was brought back to room temperature and left for 24 hours. Thetemperature of the reaction solution was further raised to 30° C., andthe reaction was continued for 2 days. The reaction solution wasfreeze-dried. To the obtained residue, 1 mL of trifluoroacetic acid wasadded under ice cooling. The mixture was brought back to roomtemperature and left for 2 hours. Subsequently, the trifluoroacetic acidwas distilled away under reduced pressure from the mixture using anevaporator. The obtained residue was dissolved by the addition of 4 mLof a 50 mM sodium acetate buffer (pH 4.0). This solution was applied ata flow rate of 1 mL/hr to SP-Sepharose HP (GE Healthcare Japan Corp.)column (1 mL) equilibrated with a 50 mM sodium acetate buffer (pH 4.0).The column was washed with 2 mL of a 50 mM sodium acetate buffer (pH4.0). Subsequently, 5 mL of a 50 mM sodium acetate buffer (pH 5.0)containing 1 M NaCl was applied to the column to obtain an elutedfraction. A glycerol skeleton-containing 2-branched urethanelinkage-type PEG (20k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(20k)-CO-^(α)NH-(h.AM (6-52))) (30):

and unreacted h.AM (6-52) peptide were recovered in the eluted fraction.This eluted fraction was concentrated into 0.2 mL using anultrafiltration membrane (Amicon Ultra 4, Merck Millipore). The obtainedconcentrate was purified and fractionated using a HPLC system (L-2000;manufactured by Hitachi High-Tech Science Corp.) connected with Tsk gelG2000SWxL (60 cm, Tosoh Corp.) column (eluent: 80 mM sodium acetatebuffer, pH 6+20% CH₃CN containing 80 mM Na₂SO₄, flow rate: 0.5 mL/min).The preparative HPLC yielded 0.2 mg (based on h.AM (6-52)) of thecompound (30) of interest.

Experiment II-1-13: Synthesis of GL-2-Branched CH₃O-PEG(20k)-CO—^(α)NH-(h.AM (11-52)) (Compound (31))

A Cys¹⁶-Cys²¹ disulfide bridge form of a peptide having the amino acidsequence ofFmoc-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “Fmoc-^(α)NH-(h.AM (11-52))”) wassynthesized by the Fmoc peptide synthesis method. 6 mg of h.AM (11-52)peptide having 4 lysine residues protected with Boc groups was obtainedas a white powder by the same procedures as in experiment II-1-12 usingFmoc-^(α)NH-(h.AM (11-52)).

The same procedures as in experiment II-1-12 were carried out exceptthat the h.AM (6-52) peptide was changed to the h.AM (11-52) peptideobtained as described above, and 20 mg of a p-nitrophenyl ester-typeCH₃O-PEGylation reagent (PEG-9) with a weight-average molecular weightof 20 kDa represented by formula (XII-1-1′):

was used, to obtain a glycerol skeleton-containing 2-branched urethanelinkage-type PEG (20k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(20k)-CO—^(α)NH-(h.AM (11-52))) (31):

Preparative HPLC yielded 0.2 mg (based on h.AM (11-52)) of the compound(31) of interest.

Experiment II-1-14: Synthesis of GL-2-Branched CH₃O-PEG(20k)-CO—^(α)NH-(h.AM (16-52)) (Compound (32))

A Cys¹⁶-Cys²¹ disulfide bridge form of a peptide having the amino acidsequence ofFmoc-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH₂(hereinafter, also described as “Fmoc-^(α)NH-(h.AM (16-52))”) wassynthesized by the Fmoc peptide synthesis method. 6 mg of h.AM (16-52)peptide having 4 lysine residues protected with Boc groups was obtainedas a white powder by the same procedures as in experiment II-1-12 usingFmoc-^(α)NH-(h.AM (16-52)).

The same procedures as in experiment II-1-12 were carried out exceptthat the h.AM (6-52) peptide was changed to the h.AM (16-52) peptideobtained as described above, and 15 mg of a p-nitrophenyl ester-typeCH₃O-PEGylation reagent (PEG-9) with a weight-average molecular weightof 20 kDa represented by formula (XII-1-1′):

was used, to obtain a glycerol skeleton-containing 2-branched urethanelinkage-type PEG (20k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(20k)-CO—^(α)NH-(h.AM (16-52))) (32):

Preparative HPLC yielded 0.2 mg (based on h.AM (16-52)) of the compound(32) of interest.

Experiment II-1-15: Synthesis of GL-2-Branched CH₃O-PEG(40k)-CO—NH-(h.AM (16-52)) (Compound (33))

The same procedures as in experiment II-1-14 were carried out exceptthat 32 mg of a p-nitrophenyl ester-type CH₃O-PEGylation reagent (PEG-9)with a weight-average molecular weight of 40 kDa represented by formula(XII-1-1′):

was used instead of the p-nitrophenyl ester-type CH₃O-PEGylation reagent(PEG-9) with a weight-average molecular weight of 20 kDa, to obtain aglycerol skeleton-containing 2-branched urethane linkage-type PEG (40k)adrenomedullin derivative (GL-2-branched CH₃O-PEG (40k)-CO—^(α)NH-(h.AM(16-52)) (33). Preparative HPLC yielded 0.15 mg (based on h.AM (16-52))of the compound (33) of interest.

Experiment II-1-16: Synthesis of GL-4-branched CH₃O-PEG(40k)-CO—^(α)NH-(h.AM (6-52)) (Compound (34))

The same procedures as in experiment II-1-12 were carried out exceptthat 20 mg of a p-nitrophenyl ester-type CH₃O-PEGylation reagent(PEG-10) with a weight-average molecular weight of 40 kDa represented byformula (XII-2-1′):

was used instead of the p-nitrophenyl ester-type CH₃O-PEGylation reagent(PEG-9) with a weight-average molecular weight of 20 kDa, to obtain aglycerol skeleton-containing 4-branched urethane linkage-type PEG (40k)adrenomedullin derivative (GL-4-branched CH₃O-PEG (40k)-CO—^(α)NH-(h.AM(6-52)) (34):

Preparative HPLC yielded 0.15 mg (based on h.AM (6-52)) of the compound(34) of interest.

Experiment II-2: Structural Analysis of N-Terminally DeletedAdrenomedullin Derivative Experiment II-2-1: Identification of BindingPosition to PEG Group by Mass Spectrometry of Cleaved Peptide

A cleaved peptide was obtained from the compounds (18), (19), (20),(21), (22), (23), (24), (29), (30), (31), (32), (33) and (34) by thesame procedures as in experiment I-2-2 using lysyl endopeptidase. Theobtained cleaved peptide was purified and fractionated by RP-HPLC by thesame procedures as in experiment I-2-2. As a result, peaks correspondingto the peaks (5), (6), (7) and (8) shown in FIG. 1 were detected in allthe RP-HPLC chromatograms of the cleaved peptides derived from thecompounds. From the results of experiment I-2-2, the peaks (1) and (5)were confirmed to correspond to the peptide fragment of h.AM (39-46),the peaks (2) and (6) were confirmed to correspond to the peptidefragment of h.AM (47-52), the peaks (3) and (7) were confirmed tocorrespond to the peptide fragment of h.AM (26-36), the peak (4) wasconfirmed to correspond to the peptide fragment of h.AM (1-25), and thepeak (8) was confirmed to correspond to a compound having a PEG groupattached to the N-terminal peptide fragment of the h.AM (1-52) peptide.From these results, therefore, all the PEG groups in the compounds (18),(19), (20), (21), (22), (23), (24), (29), (30), (31), (32), (33) and(34) were confirmed to be attached to the N-terminal α-amino groups.

Experiment II-2-2: Identification of Binding Position to PEG Group byAmino Acid Sequence Analysis

The compounds (18), (21), (22), (23), (24), (31), (33) and (34) weresubjected to amino acid sequence analysis using a protein sequencer(Procise 494 HT Protein Sequencing System, Applied Biosystems, Inc.). Asa result, an amino acid corresponding to the N-terminal amino acidresidue of human adrenomedullin was detected in none of the compounds.From these results, all the PEG groups in the compounds (18), (21),(22), (23), (24), (31), (33) and (34) were confirmed to be attached tothe N-terminal α-amino groups.

Experiment II-2-3: Molecular Weight Analysis by SDS-PAGE

In accordance with the laboratory textbook (Experimental Medicine,Suppl., “Handbook for Protein Experiments”, Yodosha Co., Ltd., edited byTadaomi Takenawa and Toshiki Ito), the compounds (15), (16), (17), (18),(19), (20), (21), (22), (23), (24), (29), (30), (31), (32), (33) and(34) (200 ng each) obtained in experiment II-1 were separated bySDS-PAGE using a polyacrylamide gel with a concentration gradient from10% to 20%. The results are shown in FIGS. 3, 4 and 5. In FIG. 3, lane 0depicts a molecular weight standard, lane 1 depicts the aforementionedcompound (1), lane 2 depicts the aforementioned compound (2), lane 3depicts the aforementioned compound (13), lane 4 depicts theaforementioned compound (14), lane 5 depicts the compound (15), lane 6depicts the compound (16), and lane 7 depicts the compound (17). In FIG.4, lane 0 depicts a molecular weight standard, lane 1 depicts theaforementioned compound (25), lane 2 depicts the aforementioned compound(26), lane 3 depicts the aforementioned compound (27), lane 4 depictsthe aforementioned compound (28), lane 5 depicts the compound (29), lane6 depicts the compound (30), lane 7 depicts the compound (31), lane 8depicts the compound (32), lane 9 depicts the compound (33), lane 10depicts the compound (34), lane 11 depicts the aforementioned compound(35), lane 12 depicts compound (36) mentioned later, and lane 13 depictscompound (37) mentioned later. In FIG. 5, lane 0 and 1 depict amolecular weight standard, lane 2 depicts the compound (18), lane 3depicts the compound (19), lane 4 depicts the compound (20), lane 5depicts the compound (21), lane 6 depicts the compound (22), lane 7depicts the compound (23), and lane 8 depicts the compound (24). All themolecular weight standards used were Precision Plus Protein™ Dual XtraStandards (Bio-Rad Laboratories, Inc.). As shown in FIGS. 3, 4 and 5,each compound was confirmed to have the desired molecular weight.

Experiment II-2-4: Confirmation of Association by Gel Filtration HPLC

Association of adrenomedullin derivative molecules was confirmed by gelfiltration HPLC using a gel filtration column (Superdex 200 Increase10/300 GL, GE Healthcare Japan Corp.). The compounds (18), (21), (22),(23), (24), (29) and (34) (50 μg each) obtained in experiment II-1 wereadded to the column. An eluent (100 mM sodium acetate and 100 mM sodiumsulfate, pH 6.0) was applied to the column at a flow rate of 0.75mL/min. From the obtained gel filtration chromatogram, each compoundexhibited a single peak having a retention time appropriate for themolecular weight. From these results, each adrenomedullin derivativemolecule was confirmed to be present as a monomer without beingassociated. The retention time of each compound in the gel filtrationchromatogram is shown in Table 2.

TABLE 2 Adrenomedullin derivative Retention time (min) Compound (18)21.4 Compound (21) 16.2 Compound (22) 16.2 Compound (23) 16.2 Compound(24) 13.9 Compound (29) 13.9 Compound (34) 14.5

Experiment III: Preparation of C-Terminally Glycine-ExtendedAdrenomedullin Derivative Experiment III-1: Synthesis of C-TerminallyGlycine-Extended Adrenomedullin Derivative Experiment III-1-1: Synthesisof GL-2-Branched CH₃O-PEG (40k)-CH₂—^(α)NH-(h.AM (1-52))-Gly (Compound(36))

The same procedures as in experiment I-1-5 were carried out except thatthe h.AM (1-52) peptide was changed to h.AM (1-52)-Gly peptide, and 80mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 40 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(40k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(40k)-CH₂—NH-(h.AM (1-52)-Gly)) (36):

Preparative HPLC yielded 0.8 mg (based on h.AM (1-52)-Gly) of thecompound (36) of interest.

Experiment III-1-2: Synthesis of GL-2-Branched CH₃O-PEG(60k)-CH₂—^(α)NH-(h.AM (1-52))-Gly (Compound (37))

The same procedures as in experiment I-1-5 were carried out except thatthe h.AM (1-52) peptide was changed to h.AM (1-52)-Gly peptide, and 80mg of an aldehyde-type CH₃O-PEGylation reagent (PEG-3) with aweight-average molecular weight of 60 kDa represented by formula(VII-1-1′):

was used instead of the CH₃O-PEGylation reagent (PEG-2), to obtain aglycerol skeleton-containing 2-branched alkylamine linkage-type PEG(60k) adrenomedullin derivative (GL-2-branched CH₃O-PEG(60k)-CH₂—^(α)NH-(h.AM (1-52)-Gly)) (37):

Preparative HPLC yielded 0.7 mg (based on h.AM (1-52)-Gly) of thecompound (37) of interest.

Experiment III-2: Structural Analysis of C-Terminally Glycine-ExtendedAdrenomedullin Derivative Experiment III-2-1: Identification of BindingPosition to PEG Group by Amino Acid Sequence Analysis

The compounds (36) and (37) were subjected to amino acid sequenceanalysis using a protein sequencer (Procise 494 HT Protein SequencingSystem, Applied Biosystems, Inc.). As a result, an amino acidcorresponding to the N-terminal amino acid residue of humanadrenomedullin was detected in neither of the compounds. From theseresults, all the PEG groups in the compounds (36) and (37) wereconfirmed to be attached to the N-terminal α-amino groups.

Experiment IV: Use Examples of Adrenomedullin Derivative ExperimentIV-1: Intracellular cAMP Concentration-Increasing Effect ofAdrenomedullin Derivative

The physiological effect of adrenomedullin (AM) is known to be exertedvia increase in the concentration of intracellular cAMP (see Non PatentLiterature 1). Accordingly, each compound prepared in experiments I-1,II-1 and III-1, full-length AM, N-terminally deleted AM, or C-terminallyglycine-extended AM was added to a cultured cell line (HEK293 cell line)caused to express an AM receptor, and the amount of intracellular cAMPproduced was measured. 10⁻⁸ mol/L of each compound, h.AM (1-52), h.AM(6-52), h.AM (11-52), h.AM (16-52), or h.AM (1-52)-Gly was added toconfluent HEK293 cells (cell count: 5×10⁴) in the presence of 0.5 mMIBMX and incubated for 15 minutes. Then, the intracellular cAMPconcentration in the HEK293 cells of each test zone was measured usingan ELISA kit for cAMP measurement (GE Healthcare Japan Corp., #RPN2251).The intracellular cAMP concentration-increasing effects of theadrenomedullin derivatives on the AM receptor-expressing cultured cellsare shown in Table 3.

TABLE 3 Intracellular cAMP concentration- Compound increasing effect(%)¹⁾  (1) 81  (2) 67  (3) 101   (4) 100   (5) 100   (6) 91  (7) 96  (8)99  (9) 94 (10) 102  (11) 95 (12) 81 (13) 80 (14) 100  (15)  101*⁾ (16)  16**⁾ (17)     9***⁾ (18) 90 (19) 80 (20) 39 (21)   98*⁾ (22) 97 (23)97 (24)    93***⁾ (25) 101  (26) 99 (27) 87 (28) 85 (29) 77 (30) 99 (31)104  (32) 96 (33) 61 (34) 83 (35) 91 (36)    96****⁾ (37)    91****⁾¹⁾Percentage (%) of the intracellular cAMP concentration obtained by theaddition of each compound with respect to the intracellular cAMPconcentration obtained by the addition of h.AM (1-52) *⁾Percentage (%)of the intracellular cAMP concentration obtained by the addition of eachcompound with respect to the intracellular cAMP concentration obtainedby the addition of h.AM (6-52) **⁾Percentage (%) of the intracellularcAMP concentration obtained by the addition of each compound withrespect to the intracellular cAMP concentration obtained by the additionof h.AM (11-52) ***⁾Percentage (%) of the intracellular cAMPconcentration obtained by the addition of each compound with respect tothe intracellular cAMP concentration obtained by the addition of h.AM(16-52) ****⁾Percentage (%) of the intracellular cAMP concentrationobtained by the addition of each compound with respect to theintracellular cAMP concentration obtained by the addition of h.AM(1-52)-Gly

As shown in Table 3, all the adrenomedullin derivatives tested exhibitedan intracellular cAMP concentration-increasing effect at the same levelas in the corresponding full-length AM, N-terminally deleted AM, orC-terminally glycine-extended AM without a linked PEG group. Therefore,the adrenomedullin derivatives having the linked PEG group are presumedto maintain bioactivity at the same level as in the parent compoundfull-length AM, N-terminally deleted AM or C-terminally glycine-extendedAM.

In comparison among the adrenomedullin derivative compound (2), compound(4), compound (6) and compound (14) which had the PEG group with thesame weight-average molecular weight (20 kDa) and the peptide moietyhaving the same amino acid sequence (h.AM (1-52)) and differed only inthe manner of linking between the PEG group and the peptide moiety, thecompound (4) and the compound (6), alkylamine linkage-type PEG (20k)adrenomedullin derivatives, exhibited a higher intracellular cAMPconcentration-increasing effect than that of the compound (2), an amidelinkage-type PEG (20k) adrenomedullin derivative. Likewise, the compound(14), a urethane linkage-type PEG (20k) adrenomedullin derivative,exhibited a higher intracellular cAMP concentration-increasing effectthan that of the compound (2), an amide linkage-type PEG (20k)adrenomedullin derivative.

In comparison among the adrenomedullin derivative compound (15),compound (16) and compound (17), or compound (18), compound (19) andcompound (20) which had the PEG group with the same weight-averagemolecular weight (5 kDa) and the peptide moiety having the same aminoacid sequence (h.AM (6-52), h.AM (11-52) or h.AM (16-52)) and differedonly in the manner of linking between the PEG group and the peptidemoiety, the compound (15), the compound (16) and the compound (17),amide linkage-type PEG (5k) adrenomedullin derivatives, exhibited asignificantly decreased intracellular cAMP concentration-increasingeffect with increase in the extent of the N-terminal deletion of thepeptide moiety. On the other hand, the compound (18), the compound (19)and the compound (20), alkylamine linkage-type PEG (5k) adrenomedullinderivatives, suppressed the influence of N-terminal deletion of thepeptide moiety on the intracellular cAMP concentration-increasingeffect.

In comparison among the adrenomedullin derivative compound (4), compound(21), compound (22) and compound (23) which had the PEG group with thesame weight-average molecular weight (20 kDa) and the same manner oflinking between the PEG group and the peptide moiety and differed onlyin the amino acid sequence of the peptide moiety, the compound (21), thecompound (22) and the compound (23), alkylamine linkage-type PEG (20k)adrenomedullin derivatives, exhibited a high intracellular cAMPconcentration-increasing effect without the influence of the N-terminaldeletion of the peptide moiety on the intracellular cAMPconcentration-increasing effect.

Experiment IV-2: Blood Pressure-Lowering Effect of AdrenomedullinDerivative

Each compound prepared in experiments I-1 and II-1 or full-length AM wasadministered in a single dose of 1 nmol/kg into the vein of each ratunder anesthesia, and change in the blood pressure of the rat wasobserved. Each 11 to 14-week-old male Wistar rat was anesthetized by theinhalation of isoflurane. After tracheotomy, inhalational anesthesia wascontrolled at an isoflurane concentration of 1.5 to 2.5% and a flow rateof 0.6 to 0.8 L/min. The right jugular vein was isolated from the rat,and a catheter tube corresponding to 26 G was inserted thereto. Next,the left carotid artery was isolated from the rat thus treated, and acatheter tube corresponding to 23 G was inserted thereto. From thecatheter tube of the right jugular vein, a physiological saline-heparinsolution (physiological saline: 100 mL; heparin: 1000 units) was infusedat 2.4 mL/hr. From this catheter tube, 1 nmol/kg of the compound (2),the compound (4), the compound (8) or h.AM (1-52) was administered in aform dissolved in physiological saline. The catheter inserted in thecarotid artery was connected to a pressure transducer. The bloodpressure before the administration of the compound (2), the compound (4)or h.AM (1-52) and the blood pressure after the administration thereofwere measured over time. The relationship between the time elapsed fromthe start of the administration of the compound (2), the compound (4),the compound (8) or h.AM (1-52) and the average blood pressure is shownin FIG. 6. FIG. 6A shows the results about the compound (2), thecompound (4) and h.AM (1-52), and FIG. 6B shows the results about thecompound (8) and h.AM (1-52). In the diagram, the ordinate depicts adifference obtained by subtracting the average blood pressure before theadministration of each drug from the average blood pressure at the timeof the administration of each drug.

As shown in FIG. 6, rapidly decreased blood pressure was observed infull-length AM (h.AM (1-52)) without a linked PEG group immediatelyafter the administration. By contrast, the rapidly decreased bloodpressure observed in h.AM (1-52) immediately after the administrationwas not observed in the adrenomedullin derivatives (Compound (2),compound (4) and compound (8)) having the linked PEG group. Therefore,the adrenomedullin derivatives having the linked PEG group are presumedto be able to suppress unwanted side effects such as rapidly decreasedblood pressure that may occur in the parent compound full-length AM.

In comparison among the adrenomedullin derivative compound (2), compound(4) and compound (8) which had the PEG group with the sameweight-average molecular weight (20 kDa) and the peptide moiety havingthe same amino acid sequence (h.AM (1-52)) and differed only in themanner of linking between the PEG group and the peptide moiety, thecompound (4) and the compound (8), alkylamine linkage-type PEG (20k)adrenomedullin derivatives, further suppressed decrease in the bloodpressure immediately after the administration, as compared to thecompound (2), an amide linkage-type PEG (20k) adrenomedullin derivative.

Experiment IV-3: Measurement Over Time of Concentration in Blood ofSubcutaneously Administered Adrenomedullin Derivative—(1)

The compound (8) prepared in experiment I-1 or full-length AM wassubcutaneously administered in a single dose of 10 nmol/kg to each rat,and time-dependent change in the concentration in blood of theadrenomedullin derivative was observed. The compound (8) or h.AM (1-52)dissolved in physiological saline was subcutaneously administered toeach 7- to 8-week-old male Wistar rat (approximately 250 g). 1 day, 7days and 10 days after the start of the administration, 50 mg ofpentobarbital was intraperitoneally administered, and 300 μL of bloodwas collected each time from the tail vain under anesthesia.Immediately, 300 μg of EDTA-2Na and 21 μg of aprotinin were added to theobtained blood sample, and the mixture was centrifuged under conditionsinvolving 10 minutes and 3000 rpm to obtain plasma. The AM concentrationin the plasma of each sample was measured by radioimmunoassay (RIA)(Kitamura K, Ichiki Y, Tanaka M et al., Immunoreactive adrenomedullin inhuman plasma. FEBS Lett., vol. 341, p. 288-90, 1994). The relationshipbetween the time elapsed from the start of the administration ofcompound (8) and the AM concentration in the plasma is shown in FIG. 7.

As shown in FIG. 7, when the compound (8) was administered, the AMconcentration in the plasma was confirmed to be 2600 pM or higher 1 daylater, 740 pM or higher 7 days later, and 280 pM or higher even 10 dayslater. On the other hand, when h.AM (1-52) was administered, the AMconcentration in the plasma was 6.7 pM 1 day later (by theadministration of the compound (8)) and 0 pM (equal to or lower thandetection sensitivity) in both the measurements performed 7 days and 10days later. The AM concentration in rat plasma is known to be typicallyon the order of 1 pM (Mori, Y. et al., Long-Term Adrenomedullin InfusionImproves Survival in Malignant Hypertensive Rats. Hypertension, 2002,vol. 40, p. 107-113.). These results demonstrated that the alkylaminelinkage-type adrenomedullin derivative of the invention is present at ahigh concentration in blood over a significantly longer period ascompared to the parent molecule adrenomedullin.

Experiment IV-4: Measurement Over Time of Concentration in Blood ofAdrenomedullin Derivative Administered in Single Dose to Jugular Vein

The compound (6) prepared in experiment I-1 or full-length AM wasadministered in a single dose of 3 nmol/kg into the vein of each ratunder anesthesia, and time-dependent change in the concentration inblood of the adrenomedullin derivative was observed. Each 8 to9-week-old male Wistar rat (approximately 300 g) was anesthetized by theinhalation of isoflurane. After tracheotomy, inhalational anesthesia wascontrolled at an isoflurane concentration of 1.5 to 2.5% and a flow rateof 0.6 to 0.8 L/min. The right jugular vein was isolated from the rat,and a catheter tube corresponding to 26 G was inserted thereto. Next,the left carotid artery was isolated from the rat thus treated, and acatheter tube corresponding to 23 G was inserted thereto. From thecatheter tube of the right jugular vein, a physiological saline-heparinsolution (physiological saline: 100 ml; heparin: 1000 units) was infusedat 2.4 ml/hr. From this catheter tube, 3 nmol/kg of the compound (6) orh.AM(1-52) was administered in a form dissolved in physiological saline.From the catheter inserted in the carotid artery, 300 μl of blood wascollected over time (1 hour, 2 hours and 4 hours after the start of theadministration). Immediately, 300 μg of EDTA-2Na and 21 μg of aprotininwere added to the obtained blood sample, and the mixture was centrifugedunder conditions involving 10 minutes and 3000 rpm to obtain plasma. TheAM concentration in the plasma of each sample was measured byradioimmunoassay (Kitamura K, Ichiki Y, Tanaka M et al., Immunoreactiveadrenomedullin in human plasma. FEBS Lett., vol. 341, p. 288-90, 1994).The relationship between the time elapsed from the start of theadministration of the compound (6) or h.AM (1-52) and the AMconcentration in the plasma is shown in FIG. 8.

As shown in FIG. 8, the compound (6) significantly prolonged bloodhalf-life as compared to h.AM (1-52). These results demonstrated thatthe alkylamine linkage-type adrenomedullin derivative of the inventionsignificantly prolongs blood half-life as compared to the parentmolecule adrenomedullin.

Experiment IV-5: Effect of Suppressing Increase in Blood Pressure inSpontaneously Hypertensive Rat (SHR Rat)—(1)

The compound (8) prepared in experiment I-1 was subcutaneouslyadministered in a single dose of 336 μg/100 μL to each spontaneouslyhypertensive rat (SHR), and the blood pressure increase-suppressingeffect of the adrenomedullin derivative was observed. Each 8-week-oldmale SHR (approximately 200 g) was fed with a high-salt diet (8% NaCl).The compound (8) was administered in a form dissolved in physiologicalsaline at the time of feeding with the high-salt diet. 100 μL ofphysiological saline was subcutaneously administered in a single dose toeach male SHR (approximately 200 g) in the same condition as above in acontrol group. The blood pressure and the pulse were measured over time(2 days before and 9 days after the administration of the compound (8)or physiological saline). The blood pressure values obtained 2 daysbefore and 9 days after the administration of the compound (8) orphysiological saline are shown in FIG. 9.

As shown in FIG. 9, increase in the blood pressure was suppressed in thecompound (8) administration group compared to the control group(physiological saline administration group). These results demonstratedthat the alkylamine linkage-type adrenomedullin derivative of theinvention has a pharmacological effect of suppressing increase in theblood pressure.

Experiment IV-6: Measurement Over Time of Concentration in Blood ofSubcutaneously Administered Adrenomedullin Derivative—(2)

The compound (27) prepared in experiment I-1 was subcutaneouslyadministered in a single dose of 10 nmol/kg to each rat by the sameprocedures as in experiment IV-3, and time-dependent change in theconcentration in blood of the adrenomedullin derivative was observed.

When the compound (27) was administered, the AM concentration in theplasma was confirmed to be 3600 pM or higher 1 day later and 120 pM orhigher 7 days later. These results demonstrated that the urethanelinkage-type adrenomedullin derivative of the invention is present athigh concentration in blood over a significantly longer period ascompared to the parent molecule adrenomedullin.

Experiment IV-7: Measurement Over Time of Concentration in Blood ofSubcutaneously Administered Adrenomedullin Derivative—(3)

The compound (37) prepared in experiment III-1 was subcutaneouslyadministered in a single dose of 30 nmol/kg to each rat, andtime-dependent change in the concentration in blood of theadrenomedullin derivative was observed. The compound (37) dissolved inphysiological saline was subcutaneously administered to each 7- to8-week-old male Wistar rat (approximately 250 g). 1 day, 2 days, 4 days,7 days and 9 days after the start of the administration, 50 mg ofpentobarbital was intraperitoneally administered, and 300 μL of bloodwas collected each time from the tail vain under anesthesia.Immediately, 300 μg of EDTA-2Na and 21 μg of aprotinin were added to theobtained blood sample, and the mixture was centrifuged under conditionsinvolving 10 minutes and 3000 rpm to obtain plasma. The AM concentrationin the plasma of each sample was measured by RIA.

When the compound (37) was administered, the AM concentration in theplasma was confirmed to be 34000 pM or higher 1 day later, 1600 pM orhigher 7 days later, and 110 pM or higher even 9 days later. Theseresults demonstrated that the alkylamine linkage-type glycine-extendedadrenomedullin derivative of the invention is present at highconcentration in blood over a significantly longer period as compared tothe parent molecule adrenomedullin.

Experiment IV-8: Effect of Suppressing Increase in Blood Pressure inSpontaneously Hypertensive Rat (SHR)—(2)

The compound (37) prepared in experiment III-1 was subcutaneouslyadministered in a single dose of 30 nmol/kg to each SHR, and the bloodpressure increase-suppressing effect of the adrenomedullin derivativewas observed. The compound (37) was administered in a form dissolved inphysiological saline to each 8-week-old male SHR (approximately 200 g).100 L of physiological saline was subcutaneously administered in asingle dose to each male SHR (approximately 200 g) in the same conditionas above in a control group. The blood pressure was measured over time(1 day before and 4 days and 9 days after the administration of thecompound (37) or physiological saline). The values of change in theblood pressure obtained 4 days and 9 days after the administration ofthe compound (37) or physiological saline with respect to the averagesystolic blood pressure on the day before the administration are shownin FIG. 10.

As shown in FIG. 10, increase in the blood pressure was suppressed inthe compound (37) administration group compared to the control group.These results demonstrated that the alkylamine linkage-typeglycine-extended adrenomedullin derivative of the invention has apharmacological effect of suppressing increase in the blood pressure.

Experiment IV-9: Pharmacological Effect on Dextran Sodium Sulfate(DSS)-Induced Colitis Model

The compound (8) was studied for its improving effect on DSS-inducedcolitis models by subcutaneous administration. The compound (8) wassubcutaneously administered to the back of each mouse. On the dayfollowing the administration (day 0), colitis model preparation wasstarted by administration of 3% DSS as drinking water for 7 days. Thedose of the compound (8) was set to 3 types of doses of 1, 5 and 25nmol/kg. Physiological saline was administered as a vehicle to a controlgroup. The body weight and the form of stool were evaluated on days 3, 5and 7 counted from the start date of the DSS administration (day 0) onthe basis of the scores shown in Table 4. The relationship between thetime elapsed from the DSS-induced colitis model preparation and thetotal score in the compound (8) administration groups and the controlgroup is shown in FIG. 11.

TABLE 4 Score Criteria Weight loss 0 Absent 1  1~5% 2 5~10% 3 10~20% Stool consistency 0 Normal 2 Loose stool 4 Diarrhea Bleeding/mucous andbloody stool 0 Normal 2 Bleeding 4 Mucous and bloody stool

As shown in FIG. 11, the 5 and 25 nmol/kg groups of the compound (8)administration groups was confirmed to exhibit significant decrease inthe scores. The decrease in the scores suggests a colitis-alleviatingeffect. A tendency to decrease the wet weight of the intestinal tractwas confirmed in the 5 nmol/kg administration group, and a tendency toincrease the length of the intestinal tract was confirmed in the 25nmol/kg administration group, as compared to the vehicle control group.These results suggested that the compound (8) has an alleviating effecton the pathological condition of the DSS colitis models under this testcondition by subcutaneous administration.

Experiment IV-10: Pharmacological Effect on2,4,6-trinitrobenzenesulfonic acid (TNBS)-Induced Colitis Model

The compound (8) was studied for its improving effect on TNBS-inducedcolitis models by subcutaneous administration. 7-week-old male Wistarrats were acclimatized for 1 week. Then, the compound (8) (1 nmol/kg) orphysiological saline was subcutaneously administered to each rat (day0). Fasting was carried out for 24 hours concurrently with thesubcutaneous administration to remove stool from the body. Colitismodels were prepared by procedures described below. The concentration ofTNBS (Nacalai Tesque, Inc.) was adjusted to 30 mg/500 μL (in a 50%aqueous ethanol solution). 50 mg of pentobarbital was intraperitoneallyadministered, and a polyethylene catheter was inserted by 8 cm from theanus under anesthesia while slowly rotated, followed by injection of 500μL of the drug solution (day 1). Then, the inverted state was maintainedfor 2 minutes. The body weight and the form of diarrhea were evaluatedevery day. 14 days later, 50 mg of pentobarbital was intraperitoneallyadministered, and blood collection from the heart and excision of thelarge intestine were carried out under anesthesia. The length and weightof the excised intestinal tract were measured and compared between thegroups. The relationship between the time elapsed from the TNBS-inducedcolitis model preparation and the body weights in the compound (8)administration group and the control group is shown in FIG. 12. In thediagram, (a) depicts the day on which the compound (8) or physiologicalsaline was subcutaneously administered and fasting was started, and (b)depicts the day on which TNBS was administered. The weights of the largeintestines in the compound (8) administration group and the controlgroup are shown in FIG. 13. The intestinal tract lengths of the largeintestines in the compound (8) administration group and the controlgroup are shown in FIG. 14.

As shown in FIG. 12, weight loss due to the development of colitis wasconfirmed in the vehicle control group, whereas the weight loss due tothe development of colitis was improved in the compound (8)administration group. As colitis is developed, the weight of the largeintestine is typically increased due to swelling at the inflammatorysite. As shown in FIG. 13, increase in the weight of the large intestinewas evidently suppressed in the compound (8) administration groupcompared to the vehicle control group. As inflammation due to colitisprogresses, the intestinal tract length of the large intestine istypically decreased. As shown in FIG. 14, decrease in the intestinaltract length of the large intestine was evidently suppressed in thecompound (8) administration group compared to the vehicle control group.From autopsy report on the large intestine, evidently fewer pathologicalchanges were also confirmed in the compound (8) administration groupthan the vehicle control group. These results suggested that thecompound (8) has an alleviating effect on the pathological condition ofthe TNBS-induced colitis models under this test condition bysubcutaneous administration. Therefore, the adrenomedullin derivative ofthe invention was found to have a therapeutic effect on colitis.

Experiment IV-11: Pharmacological Effect on Pulmonary Hypertension Model

The compound (8) was studied for its improving effect on pulmonaryhypertension models by subcutaneous administration. 3-week-old maleWistar rats (Charles River Laboratories Japan, Inc.) were purchased andacclimatized for 1 week. Then, a monocrotaline solution wassubcutaneously administered at a concentration of 60 mg/kg to each rat.At the same time therewith, the compound (8) (1 nmol/kg) orphysiological saline was subcutaneously administered in a single dose toanother position of the back. The general weight ratio between the rightventricle and the left ventricle of the heart was measured as an indexfor determining the effect on the pulmonary hypertension models. Thesemodels are known to exhibit increase in the weight ratio caused byhypertrophy of the right ventricle as the pathological conditionprogresses (Miyauchi T., Yorikane R., Sakai S., Sakurai T., Okada m.,Nishikibe M., Yano M., Yamaguchi I., Sugishita Y. and Goto k.:Contribution of endogenous endothelin-1 to the progression ofcardiopulmonary alterations in rats with monocrotaline-induced pulmonaryhypertension. Circ. Res., vol. 73, pp. 887-897, 1993). 14 days after theadministration, 50 mg of pentobarbital was intraperitoneallyadministered, and blood was collected from the inferior vena cava underanesthesia. Then, the heart was excised, and its weight was measured.The excised heart was divided into the right ventricle and the leftventricle, and their respective weights were measured. The rightventricle weight/left ventricle weight ratio was calculated. The rightventricle weight/left ventricle weight ratios in the compound (8)administration group and the control group are shown in FIG. 15.

As shown in FIG. 15, the compound (8) administration group had asignificantly lower right ventricle/left ventricle weight ratio ascompared to the vehicle control group. These results suggested that thecompound (8) has an alleviating effect on the pathological condition ofthe pulmonary hypertension models under this test condition bysubcutaneous administration.

Experiment IV-12: Pharmacological Effect on Wound Model

The compound (8) was studied for its pharmacological effect on woundmodels by subcutaneous administration. 5-week-old male BALB/c-nu/nu mice(Charles River Laboratories Japan, Inc.) were purchased and acclimatizedfor 1 week. Then, 5 mg of pentobarbital was intraperitoneallyadministered to each mouse for anesthesia. The skin was disinfectedusing ethanol for disinfection. The skin of the back was pulled withfingers with the mouse in the lateral position, and pressed and cut witha round knife for skin biopsy (disposable biopsy punch with plungersystem) from one side toward the other side on a disinfected mat fordrafting to prepare two defective injuries each having a diameter of 6mm. At the same time therewith, the compound (8) (1 nmol/kg) wassubcutaneously administered in a single dose to another position of theback. Physiological saline was administered as a vehicle to a controlgroup. A dressing was applied to cover the back including the woundsites. Change in the wound areas were observed over time. Therelationship between the time elapsed from the wound model preparationand the wound areas in the compound (8) administration group and thecontrol group is shown in FIG. 16.

As shown in FIG. 16, reduction in the wound areas proceeded faster inthe compound (8) administration group than the vehicle control group.These results demonstrated that the compound (8) has a woundhealing-promoting effect under this test condition by subcutaneousadministration.

Experiment IV-13: Pharmacological Effect on Vascular Occlusion Model

In accordance with the Morris water maze test, the compound (8) wasstudied for its pharmacological effect on learning and memory disordersin vascular occlusion model rats by subcutaneous administration. Thecompound (8) was subcutaneously administered to each rat beforeoperation for vertebral artery occlusion. The dose of the compound (8)was set to two types of doses of 1 and 10 nmol/kg. Physiological salinewas administered as a vehicle to a control group. Then, the bilateralvertebral arteries were permanently occluded under anesthesia. On thenext day, the bilateral common carotid arteries were occluded for 30minutes under anesthesia using a suture thread. Then, the suture threadwas removed, and blood flow was restarted. The date of the bilateralcommon carotid artery occlusion was defined as the date of the vascularocclusion model preparation, i.e., day 0. 9 days after the modelpreparation, the rat was caused to swim in the Morris water maze in onetrial (training). The hidden platform test was conducted at intervals of4 trials/day for 5 days from 10 days after the model preparation. On thefifth day of the hidden platform test (14 days after the modelpreparation), the probe test was conducted 1 hour after the final trial.

The Morris water maze test was conducted using an experimental apparatusdescribed below. A round pool having a diameter of 150 cm, a height of45 cm, and a water depth of 30 cm was provided. A clear colorlessplatform having a diameter of 12 cm was positioned approximately 1 cmbelow the water surface in the round pool. The water temperature of theround pool was set to 23±+1° C. Indirect lighting was disposed withinthe room having the experimental apparatus placed therein, and visualcues (calendar, ball, cube and striped sheet) for the animals werearranged therein. In the test period, this arrangement was alwaysconstant. A video tracking system (Smart, Panlab, S.L.U.) was used inmeasurement.

The hidden platform test was conducted by procedures described below. 9days after the operation, the rat was caused to swim for 90 secondswithout placing the platform and accustomed to water (training).Measurement was started from 10 days after the operation. Themeasurement was carried out at intervals of 4 trials/day. The swimmingtime to arrive at the platform from the start (escape latency) wasmeasured. The start position was changed among the trials. The positionof the platform was fixed to the same position for all the trials. Thelongest swimming time was set to 90 seconds per trial. A stay time of 30seconds on the platform after the swimming was established for a ratthat was unable to arrive at the platform within the longest swimmingtime.

The probe test was conducted by procedures described below. The pool wasdivided into 4 parts without placing the platform in the experimentalapparatus. During the hidden platform test, the swimming time in a zonehaving the platform placed therein was measured. The stay rate (%) wascalculated according to an expression given below using the measuredswimming time. The swimming time of the probe test was set to 60seconds. Only one trial was performed 1 hour after the termination ofthe final trial of the hidden platform test conducted 14 days after themodel preparation.

${{Stay}\mspace{14mu}{{rate}(\%)}} = {\frac{\mspace{14mu}\begin{matrix}{{Swimming}\mspace{14mu}{time}\mspace{14mu}\left( \sec \right)\mspace{14mu}{in}\mspace{14mu}{the}} \\{{zone}\mspace{14mu}{having}\mspace{14mu}{the}\mspace{14mu}{platform}\mspace{14mu}{placed}\mspace{14mu}{therein}}\end{matrix}}{60\mspace{14mu}\sec} \times 100}$

The relationship between the time elapsed from the vascular occlusionmodel preparation and the escape latency in the hidden platform test inthe compound (8) administration groups and the control group is shown inFIG. 17. The stay rates in the probe test in the compound (8)administration groups and the control group of the vascular occlusionmodel rats are shown in FIG. 18. As shown in FIG. 17, the 1 and 10nmol/kg administration groups of the compound (8) administration groupsshortened the time to arrive at the platform, i.e., the escape latency,in the hidden platform test as compared to the vehicle control group. Asshown in FIG. 18, the 1 and 10 nmol/kg administration groups of thecompound (8) administration groups also significantly increased the stayrate in the probe test as compared to the vehicle control group. In thistest, no significant difference in the death rate associated with theoperation for the bilateral common carotid artery occlusion wasconfirmed. These results demonstrated that the compound (8) has analleviating effect on learning and memory disorders in the 4-vesselocclusion model rats under this test condition by subcutaneousadministration.

Experiment IV-14: Pharmacological Effect on Adjuvant-Induced ArthritisModel

The compound (8) was studied for its pharmacological effect onadjuvant-induced arthritis models by subcutaneous administration. Thecompound (8) was subcutaneously administered to each rat. On the dayfollowing the administration of the compound (8) (day 1), an adjuvant(prophlogistic agent) was subcutaneously administered in a dose of 0.1mL/animal to the right hind limb of the animal to induce arthritis. Thedose of the compound (8) was set to two types of doses of 1 and 10nmol/kg. Physiological saline was administered as a vehicle to a controlgroup. The paw volumes and swelling rates of the right and left legswere measured on days 0 (day before the adjuvant administration), 4, 7,10 and 14 using a paw volume measurement apparatus (MK-550, MuromachiKikai Co., Ltd.). Inflammation scores on days 0 (day before the adjuvantadministration), 4, 7, 10 and 14 were evaluated on the basis of thescores shown in Table 5. The relationship between the time elapsed fromthe administration of the compound (8) or the vehicle and the pawvolumes exhibited after the adjuvant administration in the compound (8)administration groups and the control group is shown in FIG. 19. Therelationship between the time elapsed from the administration of thecompound (8) or the vehicle and the swelling rates exhibited after theadjuvant administration in the compound (8) administration groups andthe control group is shown in FIG. 20. The relationship between the timeelapsed from the administration of the compound (8) or the vehicle andthe inflammation scores exhibited after the adjuvant administration inthe compound (8) administration groups and the control group is shown inFIG. 21.

TABLE 5 Score Site Criteria 0 Right front limb Normal 1 Left front limbFlare/swelling in only one small joint of Left hind limb the finger 2Flare/swelling in two or more small joints or relatively large joint ofthe ankle 3 Flare/swelling in one whole limb 4 When it was confirmedthat overall swelling in one more limb reached the maximum level

As shown in FIGS. 19 and 20, the 1 and 10 nmol/kg administration groupsof the compound (8) administration groups significantly decreased thepaw volume and the swelling rate as compared to the vehicle controlgroup. As shown in FIG. 21, the 1 and 10 nmol/kg administration groupsof the compound (8) administration groups significantly decreased thearthritis score as compared to the vehicle control group. These resultsdemonstrated that the compound (8) has an alleviating effect onarthritis in the adjuvant-induced arthritis model rats under this testcondition by subcutaneous administration.

All publications, patent and patent applications cited herein areincorporated herein by reference in their entirety.

The invention claimed is:
 1. A compound represented by formula (I):A-CH₂—B  (I); or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable hydrate thereof, wherein A is a modifyinggroup represented by the following formula (V), (VI), (VII) or (VIII):

wherein a is an integer of 1 or larger; M³, M^(3′), M^(3″), M^(3′″) andM^(3″″) are each independently a bond or a polyethylene glycol grouprepresented by formula (III):^(#)—(CH₂CH₂O)_(n)—**  (III) wherein n is an integer of 1 or larger; **is a binding position to R³, R^(3′) or CH; and # is a binding positionto O, wherein the polyethylene glycol group represented by formula (III)has a weight-average molecular weight ranging from 1 to 100 kDa intotal; when a plurality of M³, M^(3′), M^(3″), M^(3′″) or M^(3″″) arepresent, the plurality of M³, M^(3′), M^(3″), M^(3′″) or M^(3″″) are thesame as or different from each other, and at least one of M³, M^(3′),M^(3″), M^(3′″) and M^(3″″) is a polyethylene glycol group representedby formula (III); R¹, R^(1′), R^(1″) and R^(1″″) are each independentlyhydrogen, substituted or unsubstituted C₁-C₂₀ alkyl, substituted orunsubstituted C₂-C₂₀ alkenyl, substituted or unsubstituted C₂-C₂₀alkynyl, substituted or unsubstituted C₃-C₂₀ cycloalkyl, substituted orunsubstituted C₄-C₂₀ cycloalkenyl, substituted or unsubstituted C₄-C₂₀cycloalkynyl, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl, substituted or unsubstituted C₇-C₂₀ cycloalkylalkyl,substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C₁-C₂₀alkyl, substituted or unsubstituted C₄-C₂₀ aryl, substituted orunsubstituted C₅-C₂₀ arylalkyl, substituted or unsubstituted 5- to15-membered heteroaryl, substituted or unsubstituted 5- to 15-memberedheteroaryl-C₁-C₂₀ alkyl, or substituted or unsubstituted acyl; R² is abond, substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted C₂-C₂₀ alkenylene, substituted or unsubstituted C₂-C₂₀alkynylene, substituted or unsubstituted C₃-C₂₀ cycloalkylene,substituted or unsubstituted C₄-C₂₀ cycloalkenylene, substituted orunsubstituted C₄-C₂₀ cycloalkynylene, substituted or unsubstituted 3- to6-membered heterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, or substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene (the groupsoptionally comprise one or more heteroatoms, an amide group (—CO—NH—),an ester group (—CO—O—), or a urethane group (—O—CO—NH—)), an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—), R³, R^(3′) and R^(3″) are each independently a bond,substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted C₂-C₂₀ alkenylene, substituted or unsubstituted C₂-C₂₀alkynylene, substituted or unsubstituted C₃-C₂₀ cycloalkylene,substituted or unsubstituted C₄-C₂₀ cycloalkenylene, substituted orunsubstituted C₄-C₂₀ cycloalkynylene, substituted or unsubstituted 3- to6-membered heterocycloalkylene, substituted or unsubstituted C₇-C₂₀cycloalkylalkylene, substituted or unsubstituted 3- to 6-memberedheterocycloalkyl-C₁-C₂₀ alkylene, substituted or unsubstituted C₄-C₂₀arylene, substituted or unsubstituted C₅-C₂₀ arylalkylene, substitutedor unsubstituted 5- to 15-membered heteroarylene, or substituted orunsubstituted 5- to 15-membered heteroaryl-C₁-C₂₀ alkylene (the groupsoptionally comprise one or more heteroatoms, an amide group (—CO—NH—),an ester group (—CO—O—), or a urethane group (—O—CO—NH—)), an amidegroup (—CO—NH—), an ester group (—CO—O—), or a urethane group(—O—CO—NH—), wherein when a plurality of R³, R^(3′) or R^(3″) arepresent, the plurality of R³, R^(3′) or R^(3″) are the same as ordifferent from each other; and * is a binding position to the othermoieties; B is a peptide moiety derived from adrenomedullin or amodified form thereof with adrenomedullin activity, wherein the peptidemoiety B is linked to the other moieties through a covalent bond of thenitrogen atom of the N-terminal α-amino group of the peptide moiety B tothe carbon atom of the methylene group thereby forming an alkylaminelinkage.
 2. The compound according to claim 1, wherein theadrenomedullin or the modified form thereof with adrenomedullin activityis a peptide selected from the group consisting of: (i) a peptideconsisting of an amino acid sequence of adrenomedullin, (ii) a peptidethat consists of an amino acid sequence of adrenomedullin and has adisulfide bond formed by two cysteine residues in the amino acidsequence, (iii) the peptide of (ii) wherein the disulfide bond of thepeptide is substituted with an ethylene group and the peptide hasadrenomedullin activity, (iv) any peptide of (i) to (iii) wherein thepeptide has the amino acid sequence comprising deletion, substitution,or addition of one to fifteen amino acid residues and has adrenomedullinactivity, (v) any peptide of (i) to (iv) wherein the peptide is amidatedat the C-terminus thereof, and (vi) any peptide of (i) to (iv) whereinthe peptide has a glycine residue added to the C-terminus thereof. 3.The compound according to claim 2, wherein the adrenomedullin or themodified form thereof is a peptide selected from the group consistingof: (i) a peptide consisting of an amino acid sequence ofadrenomedullin, (ii) a peptide that consists of an amino acid sequenceof adrenomedullin and has a disulfide bond formed by two cysteineresidues in the amino acid sequence, (v) the peptide of (i) or (ii)wherein the peptide is amidated at the C-terminus thereof, and (vi) thepeptide of (i) or (ii) wherein the peptide has a glycine residue addedto the C-terminus thereof.
 4. The compound according to claim 2, whereinthe adrenomedullin or the modified form thereof is a peptide selectedfrom the group consisting of: (iv′) any peptide of (i) to (iii) whereinthe peptide has deletion of amino acid residues at positions 1 to 15,positions 1 to 10, or positions 1 to 5 from the N-terminus thereof andhas adrenomedullin activity, (v) the peptide of (iv′) wherein thepeptide is amidated at the C-terminus thereof, and (vi) the peptide of(iv′) wherein the peptide has a glycine residue added to the C-terminusthereof.
 5. The compound according to claim 1, wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of: (a) a peptide consisting of the amino acidsequence of SEQ ID NO: 1, or a peptide consisting of the amino acidsequence of SEQ ID NO: 1 and having a disulfide bond formed by thecysteine residues at positions 16 and 21; (b) a peptide consisting ofthe amino acid sequence of the SEQ ID NO: 3, or a peptide consisting ofthe amino acid sequence of the SEQ ID NO: 3 and having a disulfide bondformed by the cysteine residues at positions 16 and 21; (c) a peptideconsisting of the amino acid sequence of SEQ ID NO: 5, or a peptideconsisting of the amino acid sequence of SEQ ID NO: 5 and having adisulfide bond formed by the cysteine residues at positions 16 and 21;(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21; (e) a peptide consisting of the amino acid sequence of SEQ IDNO: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO:9 and having a disulfide bond formed by the cysteine residues atpositions 14 and 19; (f) a peptide consisting of the amino acid sequenceof SEQ ID NO: 11, or a peptide consisting of the amino acid sequence ofSEQ ID NO: 11 and having a disulfide bond formed by the cysteineresidues at positions 14 and 19; (g) any peptide of (a) to (f) whereinthe disulfide bond of the peptide is substituted with an ethylene groupand the peptide has adrenomedullin activity; (h) any peptide of (a) to(g) wherein the peptide has the amino acid sequence comprising deletion,substitution, or addition of one to fifteen amino acids and hasadrenomedullin activity; (i) any peptide of (a) to (h) wherein thepeptide is amidated at the C-terminus thereof; and (j) any peptide of(a) to (h) wherein the peptide has a glycine residue added to theC-terminus thereof.
 6. The compound according to claim 5, wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of: (a) a peptide consisting of the amino acidsequence of SEQ ID NO: 1, or a peptide consisting of the amino acidsequence of SEQ ID NO: 1 and having a disulfide bond formed by thecysteine residues at positions 16 and 21; (b) a peptide consisting ofthe amino acid sequence of SEQ ID NO: 3, or a peptide consisting of theamino acid sequence of SEQ ID NO: 3 and having a disulfide bond formedby the cysteine residues at positions 16 and 21; (c) a peptideconsisting of the amino acid sequence of SEQ ID NO: 5, or a peptideconsisting of the amino acid sequence of SEQ ID NO: 5 and having adisulfide bond formed by the cysteine residues at positions 16 and 21;(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, ora peptide consisting of the amino acid sequence of SEQ ID NO: 7 andhaving a disulfide bond formed by the cysteine residues at positions 16and 21; (e) a peptide consisting of the amino acid sequence of SEQ IDNO: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO:9 and having a disulfide bond formed by the cysteine residues atpositions 14 and 19; (f) a peptide consisting of the amino acid sequenceof SEQ ID NO: 11, or a peptide consisting of the amino acid sequence ofSEQ ID NO: 11 and having a disulfide bond formed by the cysteineresidues at positions 14 and 19; (i) any peptide of (a) to (f) whereinthe peptide is amidated at the C-terminus thereof; and (j) any peptideof (a) to (f) wherein the peptide has a glycine residue added to theC-terminus thereof.
 7. The compound according to claim 5, wherein theadrenomedullin or the modified form thereof is a peptide selected fromthe group consisting of: (h′) any peptide of (a) to (d) wherein thepeptide has deletion of amino acid residues at positions 1 to 15,positions 1 to 10, or positions 1 to 5 from the N-terminus thereof andhas adrenomedullin activity, or the peptide of (e) or (f) wherein thepeptide has deletion of amino acid residues at positions 1 to 13,positions 1 to 8, or positions 1 to 5 from the N-terminus thereof andhas adrenomedullin activity; (i) the peptide of (h′) wherein the peptideis amidated at the C-terminus thereof; and (j) the peptide of (h′)wherein the peptide has a glycine residue added to the C-terminusthereof.
 8. A method for treating a disease, associated withintracellular cAMP, by administering an effective amount of the compoundaccording to claim 1 or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable hydrate thereof, to a subject in needthereof, wherein the disease is selected from the group consisting ofcardiovascular disease, an inflammatory disease, or a peripheralvascular disease, and wherein said administering increases theconcentrations of intracellular cAMP.
 9. The compound according to claim1, wherein A is a modifying group represented by the following formula(V-1-1), (VI-1-1), (VII-1-1), (VII-1-2), (VII-2-1), or (VIII-1-1):

wherein n′ is an integer of 1 or larger; and * is a binding position tothe other moieties.
 10. The compound according to claim 1, wherein A isa modifying group represented by formula (VI).
 11. The compoundaccording to claim 1, wherein the compound is a lysineskeleton-comprising 2-branched alkylamine linkage-type PEG (40k)adrenomedullin derivative (Lys-2-branched CH₃O-PEG(40k)-CH₂-^(α)NH-(h.AM (1-52))) having the formula (10):

wherein h.AM (1-52) is a peptide corresponding to amino acid residues 1to 52 of human adrenomedullin (SEQ ID NO: 1).
 12. A pharmaceuticalcomposition comprising the compound of claim 1, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable hydratethereof; and one or more pharmaceutically acceptable carriers.
 13. Apharmaceutical composition comprising the compound of claim 11, or apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable hydrate thereof; and one or more pharmaceutically acceptablecarriers.
 14. A method for treating a disease, associated withintracellular cAMP, by administering an effective amount of the compoundaccording to claim 11 or a pharmaceutically acceptable salt thereof, ora pharmaceutically acceptable hydrate thereof, to a subject in needthereof, wherein the disease is selected from the group consisting ofcardiovascular disease, an inflammatory disease, or a peripheralvascular disease, and wherein said administering increases theconcentrations of intracellular cAMP.